KR20220055399A - Self-transcribing RNA/DNA system that provides mRNAs in the cytoplasm - Google Patents

Abstract

The present invention relates to a self-transcription RNA/DNA system including a DdRp RNA expression cassette, and a DNAi expression cassette amplified by the DdRp, or to a RNA/DNA system including an auto_DdRp expression cassette in addition to the self-transcription RNA/DNA system. More specifically, the present invention relates to the self-transcription RNA/DNA system in which mRNAi is expressed continuously for a long period of time by increasing the expression of DdRp in the cytoplasm.

Description

RNA/DNA system that provides mRNA capable of self-transcription in the cytoplasm {Self-transcribing RNA/DNA system that provides mRNAs in the cytoplasm}

The present invention relates to a system having a component capable of synthesizing at least one endogenous protein in a cell and having a component capable of transcription, wherein the system expresses DdRp RNA including DNA dependent RNA Polymerase (DdRp) mRNA having a structure that enables intracellular translation cassette; and a DNAi expression cassette encoding the mRNA of interest (mRNAi) amplified by the DdRp; an auto_DdRp expression cassette comprising a self-transcribed RNA/DNA system or a DNA encoding the DdRp in addition to the self-transcribed RNA/DNA system It is a technology related to a self-transcribed RNA/DNA system comprising a self-transcribed RNA/DNA system that increases the expression of DdRp in the cytoplasm to continuously express mRNAi for a long period of time.

Gene therapy and genetic vaccination are among the most promising and rapidly developing methods of modern medicine. They are specific and can provide individual options for the treatment of a wide variety of diseases. In particular, not only inherited genetic diseases, but also autoimmune diseases, infectious diseases, cancerous or tumor-associated diseases as well as inflammatory diseases are of these therapeutic approaches. can be a target It is also envisaged to prevent the (early) onset of such diseases by these approaches.

A rational basis for the main concept of gene therapy is the appropriate regulation of impaired gene expression associated with the pathological condition of a particular disease. Pathologically altered gene expression may be due to insufficient or overproduction of essential gene products, for example signaling factors such as hormones, housekeeping factors, metabolic enzymes, structural proteins etc. Altered gene expression may be due to mis-regulation of transcription and/or translation, as well as mutations in ORFs encoding specific proteins. Pathological mutations can occur, for example, by chromosomal aberrations, or by more specific mutations such as point mutations or structure-shift mutations, all of which potentially result in limited function and total loss of function of the gene product. can occur However, misregulation of transcription and/or translation can also occur, and if the mutation affects the gene encoding the protein, it is involved in the transcription or translation machinery of the cell. Such mutations can lead to pathological up- or down-regulation of the gene. A gene encoding a gene product exerting such a regulatory function may be, for example, a transcription factor, a signal receptor, a messenger protein, and the like. However, the loss of function of the genes encoding these regulatory proteins is, in certain circumstances, reversed by the artificial introduction of other factors acting further downstream of the damaged gene product. These genetic defects can also be compensated for by gene therapy through replacement of the affected gene itself.

Gene therapy and genetic vaccination are essentially based on the administration of nucleic acid molecules to a patient and subsequent transcription and/or translation of coded genetic information. In gene therapy or genetic vaccination, RNA as well as DNA can be used as nucleic acid molecules for administration. DNA is known to be relatively stable and easy to handle. However, the use of DNA carries the risk of unwanted insertion of the administered DNA-fragment into the patient's genome, potentially resulting in loss of function of the damaged gene.

An additional risk is the appearance of unwanted production of anti-DNA antibodies. Another problem is the limited expression level of the encoded peptide or protein that can be achieved by DNA administration and subsequent transcription/translation thereof. Among other factors, the presence of specific transcription factors that regulate DNA transcription has a major impact on the expression level of the administered DNA. In the absence of these factors, DNA transcription cannot yield satisfactory amounts of RNA. As a result, the level of translated peptide or protein obtained is limited.

By using RNA instead of DNA for gene therapy or genetic vaccination, the risk of unwanted genomic integration and generation of anti-DNA antibodies can be minimized or completely avoided. However, RNA is considered a rather unstable molecular class. On the one hand, in the extracellular space, RNA can be degraded by the almost ubiquitous RNasi. On the other hand, the in vivo mRNA half-life in the cytoplasm is limited by the rate of enzymatic mRNA degradation, depending, at least in part, on cis-acting elements in the mRNA molecule. In doing so, the regulated degradation of mRNA contributes to good regulation of eukaryotic gene expression. Thus, each naturally occurring mRNA has its individual half-life depending on the gene from which the mRNA is derived.

For several years, it was generally accepted that mRNA was too unstable to be used effectively for gene therapy targets. However, in the past decade, not only has the potential of mRNA-mediated transformation with surprising results related to transformation efficiency and sustained protein expression been demonstrated, but also major advantages have been demonstrated through the use of pDNA. One of these advantages is the fact that mRNA does not need to cross the nuclear barrier for the synthesis of the encoded protein.

For gene therapy and genetic vaccination, modified stabilized RNA is usually more suitable than rapidly degraded unmodified_RNA. In addition, the product encoded by the RNA-sequence is advantageous for accumulation in vivo. On the other hand, RNA must maintain its structural and functional integrity when prepared into an appropriate dosage form, when administered, and during its storage. Significant efforts are needed to provide stable RNA molecules for gene therapy or genetic vaccination to prevent their premature degradation or breakdown.

After introduction into cells, the half-life of (non-stabilizing) mRNA is limited. Consequently, the production of proteins encoded by these mRNAs is maintained for up to several days. Therefore, this fact limits the application of (non-stabilizing) mRNA-based gene therapy. Because it requires repeated administration, it cannot be used, for example, to treat genetic disorders.

mRNA is generally considered significantly unstable compared to DNA, especially once it reaches the cytoplasm exposed to degrading enzymes. The main reason for its instability is the hydroxyl group on the second carbon atom of the sugar component, which prevents mRNA from forming a stable double β-helix structure with sterical hindrance, but it hydrolyzes RNA molecules better make it possible Intracellular mRNA delivery was skeptical, mainly due to the belief that mRNA was very unstable and could not withstand the transformation protocol.

One of the advantages of RNA is that the action of RNA is sufficient to deliver RNA into the cytoplasm of the cell and perform its function in the cytoplasm (as opposed to DNA, which must pass through the nuclear membrane). However, naked nucleic acid molecules do not enter cells effectively because of their large size and hydrophilic nature due to the anionically charged phosphate groups. Additionally, they are very sensitive to nuclease-mediated degradation. Therefore, the challenge for gene therapy is to develop effective and safe means of RNA delivery into cells.

In general, viral and non-viral delivery methods have been developed. With regard to safety and process economy, non-viral methods are usually preferred. Some of these methods are based on physical blockade of cell membrane barrier function, while others use cationic carrier molecules to facilitate gene introduction into target cells without degradation of the delivered gene.

As is clear from experiments using RNA as a vaccine, protein expression mediated by the introduction of heterologous mRNA in vivo is generally feasible and sufficient to elevate a detectable immune response. However, raising an effective immune response and, moreover, achieving a therapeutic effect by mRNA-mediated protein supply may be more demanding in terms of the level required for protein expression.

However, synthetic RNA has a problem with a very short duration of about 2-4 days, because RNA is easily degraded by various nucleases in the cytoplasm and the concentration of RNA is diluted when cell division occurs. Due to this short persistence problem, there is a problem that synthetic RNA needs to be injected frequently, and also has a limitation that the efficiency is very low because the persistence is too short to synthesize and provide a target protein having a long half-life. They are slowing the development of therapeutics using RNA.

Effective delivery of an RNA/DNA system in which RNA and DNA are mixed into cells of an individual thus still appears as a bottleneck in the effective expression of proteins introduced into cells by heterologous nucleic acids. The therapeutic effect of nucleic acid-based medicaments depends largely on the effective expression of gene products encoded in therapeutic nucleic acid molecules, particularly in the field of gene therapy.

Existing efforts to improve the persistence of protein synthesis of interest have been shown to be attempts to deliver as much RNA as possible by increasing the RNA loading efficiency using various carriers. Although carriers based on lipofectin, lipofectamine, cell pectin, cationic phospholipid nanoparticles, cationic polymers, or liposomes have been mainly used, carriers using cationic molecules or synthetic polymers have low transport efficiency into cells and The problem of cytotoxicity that can be induced in the process of gene transfer into the blood was pointed out.

In addition, in the case of a viral vector in which the continuity of the target protein synthesis can be exhibited for a long time, the problem that stability in the body is not ensured has been pointed out by causing immune side effects due to the immunogenicity of the surface protein of the viral vector despite its excellent persistence. Above all, due to the risk of a foreign gene being inserted into a patient's genome, target protein synthesis using viruses had to have limitations in human application.

Meanwhile, as an effort to improve the persistence of protein synthesis of interest, development of an expression cassette DNA capable of RNA expression based on a nuclear-dependent promoter is known. The RNA polymerase Ⅲ (Pol Ⅲ) promoter derived mainly from U6 small nuclear RNA and H1 RNA genes is the most utilized. We provide a way to produce it. However, this plasmid DNA method, which relies on nuclear membrane penetration, which is known to have an efficiency of 1% compared to cell membrane penetration, has very low target protein synthesis efficiency. In particular, in vivo applications, where the transfer efficiency of plasmid DNA is significantly lower, application is limited due to low efficiency. In addition, in the case of a high concentration of RNA expressed in the nucleus, it can be converted into mature RNA that can participate in target protein synthesis only when it is released into the cytoplasm, but the exportin-5 transporter involved in the release into the cytoplasm has a high concentration It causes a problem of saturation by the RNA of RNA, which prevents even the cytoplasmic ejection of RNA involved in other intracellular functions. In addition, nuclear-dependent RNA expression methods using various expression cassette DNAs are currently known, but the in vivo model still has the limitation of short persistence.

As a result of earnest efforts to develop a composition capable of continuously maintaining RNA expression in the cytoplasm, the present inventors have produced a DdRp RNA expression cassette comprising a DNA dependent RNA Polymerase (DdRp) mRNA having a structure that enables intracellular translation; And a composition comprising a DNAi expression cassette encoding mRNAi, or a composition comprising an auto_DdRp expression cassette comprising DNA encoding DdRp in addition to the composition, and a carrier comprising the same can express mRNAi continuously for a long period of time in the cytoplasm, , In particular, it was confirmed that the expression of the target gene can be selectively suppressed in cancer tissue, thereby completing the present invention.

The present invention is a system having a component capable of synthesizing at least one endogenous protein in a cell and capable of transcription, wherein the system is a DNA dependent RNA Polymerase comprising a chemically modified nucleotide (CM) having a structure that enables intracellular translation. (DdRp) DdRp RNA expression cassette comprising mRNA; and a self-transcription RNA/DNA system comprising a DNAi expression cassette comprising a DNAi encoding an mRNA of interest (mRNAi) amplified by the DdRp or auto_DdRp expression comprising a DNA encoding the DdRp in addition to the RNA/DNA system It relates to a self-transcribed RNA/DNA system comprising a cassette, which increases the expression of DdRp in the cytoplasm to continuously express mRNAi for a long period of time.

Additionally, the present invention relates to a method of providing a 5'Cap structure at the 5' end of a transcript for stability and translation efficiency of the transcript produced by DdRp, a product of the self-transcribed RNA/DNA system of the present invention. .

An object of the present invention is to increase the expression of genetic information contained in a nucleic acid molecule introduced into a cell or tissue.

In particular, it is an object of the present invention to increase the expression of the encoded protein in the mRNAi molecule administered to a subject, as well as in gene therapy or genetic vaccination.

The fundamental object of the invention is solved by the claimed subject matter.

The present invention relates to a DdRp RNA expression cassette comprising a DNA dependent RNA Polymerase (DdRp) mRNA (capped CM_DdRp mRNA) with a possible structure enabling intracellular translation; and a self-transcribed RNA/DNA system comprising a DNAi expression cassette comprising a DNA encoding an mRNA of interest (mRNAi) amplified by the DdRp or an auto- comprising DNA encoding the DdRp in addition to the RNA/DNA system It relates to a self-transcribed RNA/DNA system comprising a DdRp expression cassette and characterized in that it increases the expression of DdRp in the cytoplasm in a nuclear-independent manner to continuously express mRNAi for a long period of time.

The present invention provides a method for providing a 5'Cap structure to a transcript for stability and translation efficiency of an entire transcript produced by DdRp, a product of the auto_DdRp expression cassette of the present invention.

The present invention relates to a DNA of interest ("SM_DNAi") comprising at least one open reading frame (ORF) or at least one sequence modification (SM) that increases the expression of the encoded protein for medical use, and It provides a DNAi expression cassette including the SM_DNAi fragment.

In the RNA/DNA system of the present invention, a pharmaceutical composition and kit of parts for the DNAi or an expression cassette comprising the same are provided, preferably for use in the field of gene therapy and/or genetic vaccination.

The RNA/DNA system of the present invention provides a method for increasing the expression of a protein of interest with a capped CM_DdRp mRNA or a capped CM/SM_DdRp mRNA expression cassette and a DNAi expression cassette encoding SM_mRNAi.

The object of the present invention is solved by the provision of an RNA/DNA system, wherein the expression of the encoded protein in a target tissue or target cell can be further increased.

The following definitions are provided for clarity and readability. Any technical feature recited for these definitions may be understood in each and all of the present invention. Further definitions and explanations may be provided in particular in these. Where nucleic acid sequences are reported herein, these sequences generally include both a specific RNA or DNA sequence as well as its corresponding DNA or RNA counterpart, respectively. For example, where a DNA sequence is provided, the skilled person knows that the corresponding RNA sequence is obtained by thymine exchange with uracil residues and vice versa.

Cells : Cells include cells from any organism suitable for genome editing. Exemplary organisms include, but are not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, cats; poultry such as chickens, ducks and geese; plants such as monocotyledonous plants and dicotyledonous plants such as rice, corn, wheat, sorghum, barley, soybean, peanut, Arabidopsis, and the like.

Gene therapy : Gene therapy generally refers to the treatment of a patient's body or an isolated element of the patient's body, eg, isolated tissues/cells, by means of a nucleic acid encoding a peptide or protein. This generally involves a) administering nucleic acids, preferably RNA and DNA molecules as defined herein - by any route of administration - directly to the patient or in vivo/ex vivo or in vitro. administration to isolated cells/tissues of a patient in vitro that results in transformation of the patient's cells in vitro; b) transcription and/or translation of the introduced nucleic acid molecule; and optionally) re-administration of the isolated, transformed cells to the patient if the nucleic acid was not directly administered to the patient.

Cassette : Expression cassette refers to a vector suitable for expressing a target nucleotide sequence in an organism, such as a recombinant vector. Expression refers to the production of a functional product. For example, expression of a nucleotide sequence may refer to transcription (eg, transcription to produce mRNA or functional RNA) of the nucleotide sequence and/or translation of the RNA into a protein precursor or mature protein. The expression cassette may be a linear nucleic acid fragment, a circular plasmid, a viral vector, or a translatable RNA (eg, mRNA). An expression cassette may comprise a subject nucleotide sequence and a regulatory sequence, which may be derived from different sources, or a subject nucleotide sequence and a regulatory sequence, which are derived from the same source but aligned in a manner different from that normally found in nature.

Upstream: refers to the relative position of a first element of a nucleic acid molecule to a second element of the nucleic acid molecule, wherein the two nucleic acid molecules are comprised in the same nucleic acid molecule, wherein the first element is located 5' end of the second element of the nucleic acid molecule do. The second element is then referred to as being “downstream” of the first element of the nucleic acid molecule in question. An element located "upstream" of a second element may be synonymous with being "5'" of that second element. For double-stranded nucleic acid molecules, indications such as "upstream" and "downstream" are given for the (+) strand.

Regulatory sequences or regulatory factors: used interchangeably, upstream (5'UTR), within or downstream (3'UTR) of a coding sequence, affecting the transcription, RNA processing or stability or translation of the combined coding sequence. refers to the nucleotide sequence in which it is located. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

Promoter: refers to a nucleic acid fragment capable of controlling the transcription of other nucleic acid fragments. In the present invention, a promoter is a promoter capable of controlling the transcription of a gene in a cell, whether or not derived from the cell. The promoter may be a constitutive promoter or a tissue-specific promoter or a developmentally-regulated promoter or an inducible promoter. The core promoter refers to a nucleic acid sequence included in the promoter. A core promoter is typically the smallest portion of a promoter necessary to properly initiate transcription. A core promoter typically includes a transcription initiation site and a binding site for RNA polymerase.

Polymerase : generally refers to a molecular entity capable of catalyzing the synthesis of polymer molecules from monomeric building blocks. An “RNA polymerase” is a molecular entity capable of catalyzing the synthesis of an RNA molecule from ribonucleotide building blocks. A “DNA polymerase” is a molecular entity capable of catalyzing the synthesis of a DNA molecule from deoxyribonucleotide building blocks. In the case of DNA polymerases and RNA polymerases, the molecular entity is typically a protein or an aggregate or complex of a plurality of proteins. Typically, a DNA polymerase synthesizes a DNA molecule based on a template nucleic acid, typically a DNA molecule. Typically, RNA polymerase synthesizes an RNA molecule based on a template nucleic acid, the template nucleic acid being either a DNA molecule (where the RNA polymerase is a DNA-dependent RNA polymerase, DdRP) or an RNA molecule (where the RNA polymerase is an RNA-dependent RNA) for polymerase RdRp).

DNA dependent RNA Polymerase (DdRp) : DdRp is a synonym for RNA Polymerase (RNAP), and DdRp in the nucleus of eukaryotes is RNAP I, RNAP II and RNAP III, and they are structurally mutually exclusive. They are very different and each transcribe a different set of genes (different polymerases are present in mitochondria and chloroplasts). However, all three have two large subunits related to each other and the two largest subunits of bacterial RNAP. There are also several small subunits found in common in all three of these enzymes or only between RNAP I and RNAP III.

Eukaryotic DdRp, like bacterial RNAP, requires specialized cofactors for transcription initiation. However, unlike in the case of bacteria, eukaryotic initiators recognize promoter elements independently and not as part of the polymerase holoenzyme. Many different initiators are involved, particularly in genes transcribed by RNAP II, some of which are themselves very complex proteins. Purified eukaryotic RNA polymerase alone cannot initiate transcription selectively at the promoter.

Holoenzyme is sometimes used to refer to eukaryotic RNAP, but in this case it refers to something similar to a bacterial 'core' enzyme and cannot be transcribed from a promoter. However, unlike bacterial core enzymes, eukaryotic whole enzymes can contain many other proteins involved in the transcription or processing of RNA.

RNAP I is found in the nucleolus (phosphorus) and transcribes only the gene encoding the large ribosomal RNA from which most cellular RNA is synthesized. In yeast, the enzyme has 13 subunits (and a mass of nearly 600 kDa). Five of the smaller subunits are also found in yeast RNAP II and III and the remaining two in yeast RNAP III.

RNAP II transcribes the gene that encodes a protein, most of the genes in the cell. It also transcribes the genes that code for most small nuclear RNAs (snRNAs). Most organisms appear to have a 12-subunit RNAP II (mass about 550 kDa). However, several other proteins are required for full activity, and RNAP holoenzymes can have a mass of 4000 kDa.

RNAP III mainly transcribes genes encoding transfer RNA and 5S RNA, but also transcribes some genes encoding other small RNAs. RNAP III has at least 14 distinct subunits with a mass of nearly 700 kDa. The promoters for RNAP I and RNAP II are mostly upstream of the transcription start site (as is the case with prokaryotic promoters), but some promoters for RNAP III are downstream of the start site.

The entire kidney complex formed by these enzymes resembles bacterial RNAP. The mechanism by which these enzymes find a promoter is quite different from that used by bacteria, but the overall mechanism of transcription initiation is very similar. However, little is known about the termination of eukaryotes.

DdRp range in subunit complexity from single subunit polymerases of bacteriophages (bacterial viruses) to complexes of 12-15 subunits in eukaryotes (Table 1).

Many phages utilize the host cell's polymerase and a self-encoding RNA polymerase, as is the case with phage encoding smaller single-subunit RNA polymerases (eg T7). Some of these polymerases are currently biotechnological because they are relatively easy to mass-produce in bacterial cells, do not require additional protein cofactors to recognize the promoter region, and synthesize transcripts at least twice as fast as bacterial host RNA polymerases. used as the main reagent for Interestingly, single subunit bacteriophage RNA polymerase autologous recognizes the correct gene and purified DNA, whereas multi-subunit eukaryotic nuclear polymerase requires a very large number of accessory protein factors to find the proper location of the template for transcription. You can start at the right place in

Bacterial RNA polymerase contains four subunits of three non-identical proteins in the catalytic core of the enzyme, and this core complex recognizes specific sequences that promote transcription and adds one that reduces binding to non-promoter sites. It requires a cofactor (called sigma). Each species of bacteria has several sigma factors.

Subunit of DNA-dependent RNA polymerase Source Number of subunits in core Sizi (kDa) Bacteriophage T3, T7, <SP6 ^a One 100 Bacteriophage N4 ^a,b One 320 3 40, 30, 15 Eubacteria, for example, icherichia coli 4 150, 145, 45 Archaebacteria, for example, Sulfolobus acidocaldarius 13 122, 101, 44, 30, 27, 13.8, 12, 11.8, 10, 9.7, 9.7, 7.5, 5.5 Saccharomyci cerevisiae RNAP I 14 190, 135, 49, 43, 40, 34.5, 27, 23, 19, 14.5, 14, 12.2, 10, 10 Saccharomyci cerevisiae RNAP II 12 191, 140, 35, 25.4, 25, 19, 17.9, 16.5, 14.2, 13, 8.3, 7.7 Saccharomyci cerevisiae RNAP III 13-17 160, 128, 82, 53, 40, 37, 34, 31, 27, 25, 23, 19, 17, 14.5, 11, 10, 10 Human and yeast mitochondria One 140 Spinach and mustard chloroplast ^c One 110 4 154, 120, 78, 38 13 141, 110, 36, 26, and others not yet fully characterized Vaccinia virus 8 147, 132, 35, 30, 22, 19, 18, 7 Baculovirus ^a 4 98, 55, 53, 46

a, Viruses use host RNA polymerase as well as self-encoding RNA polymerase for temporal regulation of transcription. b, Virus encodes one or more RNA polymerases.

c, One or more RNA polymerases are used.

The genomes of some bacteriophages, such as Bacillus subtilis phage SP01 and E. coli phage T4, encode autologous sigma factors that redirect the bacterial RNA polymerase catalytic core for transcription of the viral DNA template. The catalytic core of bacterial enzymes is used equally for all these transcriptions. In addition, many bacteriophages utilize other viral-coding and host-coding regulatory proteins to modify subsequent steps of the transcription cycle after promoter recognition.

Bacteriophage N4 uses both a host polymerase and the other two polymerases encoded by its own genome (Table 1). One of the viral polymerases is a very large single-subunit enzyme, which, unlike the smaller single-subunit bacteriophage-encoding polymerases, requires a single-stranded DNA binding protein, the host cofactor ssb. The ssb protein allows this 320 kDa polymerase to recognize a specific hairpin structure in viral DNA. This allows the synthesis of additional viral RNA, allowing for the replication and assembly of new viral particles.

Like prokaryotic viruses, eukaryotic viruses use a full spectrum of transcriptional strategies, from coding their own polymerases (eg, vaccinia, baculovirus) to using host cell machinery. Many eukaryotic viruses also encode RNA-dependent RNA polymerase (RdRp) (eg hepatitis C virus, polio virus). Virus-encoded protein factors also regulate host cell transcription at various locations in the transcriptional cycle.

Eukaryotic DdRp can be found in the nucleus and organelles. A polymerase found in mitochondria and one polymerase found in chloroplasts resemble small single-subunit bacteriophage RNA polymerases. There are two other RNA polymerases in chloroplasts, one of which is very similar to bacterial enzymes (Table 1). Nuclear RNA polymerase is provided as three distinct biochemically isolated complexes, each originally classified according to the type of gene it transcribes. More recently, it has been defined by subunits and accessory protein factors that act to regulate RNA synthesis. Termed RNA polymerases I, II and III (or A, B and C, respectively) each have at least 12 subunits (Table 1). RNA polymerase I transcribes a gene encoding structural RNA for a subunit of the ribosome. RNA polymerase II transcribes genes encoding a subset of proteins and small RNAs. RNA polymerase III transcribes genes encoding a subset of ribosomal 5S RNA, tRNA, and other small RNAs.

Transcription by DdRp : DNA-dependent RNA polymerases of the bacteriophage T3, T7 or SP6 are a family of relatively small (~100 kDa) single-subunit RNA polymerases that are responsible for all stages of transcription, i.e. initiation, elongation or termination. No additional protein factors are required. These polymerases are easily overexpressed in E. coli. They are very active, terminate less frequently (compared to E. coli RNA polymerase) and transcribe very tightly in their promoters. These properties make these polymerases a very convenient tool for in vitro RNA synthesis using various experimental strategies. RNA polymerase from the bacteriophage T7 (T7 RNAP) is perhaps the most commonly used and commercially available, but more economical to purify in-house for large-scale RNA synthesis.

As shown in <Figure 1>, in general, bacteriophage RNAP starts and terminates RNA transcription at a specific sequence (with a specific efficiency). However, the simplest experimental setup for the production of ready RNA in vitro is the so-called efflux transcription, in which RNA synthesis begins at a specific T7 promoter and ends when RNAP detaches from the physical end of the DNA template. This setup allows for the convenient preparation of chemically synthesizeable DNA promoter and template sequences. Moreover, it has been found that the DNA template need not be perfectly base paired. Most or all of the coding strand may remain single stranded. The minimum base pair region spanning positions -15 to -3 (+1 indicates the start of the transcription residue) is still sufficient for fully efficient transcription using T7 RNAP. This is consistent with the notion of forming a transcriptional bubble as part of the promoter recognition process. In the crystal structure of T7 RNAP with an open promoter, the template and non-template strands are unwound downstream starting at position -4. This property makes it possible to prepare a single universal DNA top strand for transcription of any RNA sequence. Only the bottom coding DNA strand needs to be redesigned each time.

As shown in Figure 1, the native T7 RNAP promoter sequence is strongly conserved from position -17 to position +6. Nevertheless, RNA is transcribed even in promoters chemically modified at positions +1 to +6, although yields may be reduced. In general, the most efficient yields are achieved with RNA sequences beginning with GG or GA. To improve the yield, the transcription reaction conditions should be optimized for each RNA sequence. These include varying MgCl2 concentrations and relative amounts of T7 RNAP, template and NTP. In addition to the correct RNA fragment and several shorter stop products, in vitro transcription, T7 RNAP, often incorporates a non-template nucleotide at the 3' end of the main transcript to generate the so-called 'n+1 product'. The desired RNA product must then be isolated from mis-sized transcripts, DNA templates, and unused NTPs and RNAPs.

As shown in <Fig. 1>, RNA having almost any length and sequence can be prepared through in vitro transcription using T7 RNAP. However, for larger RNAs, the use of chemically synthesized DNA templates is not practical because the yield of templates with oligonucleotide length decreases exponentially. For templates larger than 50-100 nt, an alternative approach is to use a fully double-stranded DNA template designed within a linearized high-copy DNA plasmid (eg pUC18.31). Another potential complication of preparing large RNAs is RNA denaturation. This is done during PAGE purification. RNA has to be folded back into its original form after such purification, which can sometimes be problematic.

DdRp promoter : DdRp encoded by bacteriophages T7, T3 and SP6 is particularly suitable for polymerase-promoter interaction studies. Each phage enzyme consists of a single species of protein = 100 kDa capable of carrying out all steps of the transcription process in the absence of additional protein factors. Bacteriophage T7 is a prototype of a group of bacterial viruses, each encoding a single subunit DdRp. Other members of this class include coliphagi coli phage T3 and BA14, Salmonella phage Salmonella phage SP6, Pseudomonasphage Pseudomonas phage gh-1 and Klebsiella phage phage K11. Phage DdRp is useful in a variety of applications, including large-scale RNA synthesis, nucleic acid amplification, and has proven to be the basis of efficient expression systems in both prokaryotic and eukaryotic cells.

As shown in <Fig. 2>, despite the high level of structural similarity (judging from the conservation of amino acid sequence), phage DdRp acts precisely and specifically for its promoter sequence. Each phage promoter is associated with a consensus 23 base pair consensus sequence that extends from -17 to +6. The core nucleotide sequence, extending from -7 to +1, is highly conserved across the three promoter types, suggesting that these regions interact in a common manner with the shared features of polymerases. Promoter sequences differ significantly in the region from -8 to -12, suggesting that base pairing in this region is important for specific promoter recognition. Studies using synthetic phage promoters have shown that the key determinants of promoter specificity for T3 and T7 enzymes include the base pairs at positions -10 and -11. Substitution of the T3 residue at these two positions in the T7 promoter sequence prevents recognition by T7 polymerase and enables transcription by T3 RNA polymerase. The domains of the T3 and T7 RNA polymerases responsible for the recognition of these determinants were localized, and preliminary crystallographic data suggest that this region of the polymerase is located within the putative DNA binding cleft.

Nucleic Acid Molecules : A nucleic acid molecule is a molecule that preferably comprises, preferably consists of, a nucleic acid component. The term nucleic acid molecule refers to either DNA or RNA. It is used synonymously with the term "polynucleotide". Nucleic acid molecules are polymers comprising or consisting of nucleotide monomers and are covalently linked to each other by phosphodiester bonds of sugar/phosphate-backbone.

DNA: DNA is a generic abbreviation for deoxyribonucleic acid. It is a nucleic acid molecule, ie a polymer made up of nucleotides. These nucleotides usually consist of - by themselves - a sugar moiety, a base component and a phosphate component: deoxyadenosine-monophosphate, deoxythymidine-monophosphate, deoxyguanosine-monophosphate and between deoxy It is a tidine-monophosphate monomer and is polymerized by a unique backbone structure. The backbone structure is generally formed by a phosphodiester bond between the sugar moiety of the first nucleotide proximal to the monomer, i.e., deoxyribose and the second phosphate moiety. The specific order of the monomers, ie the order of the bases linked to the sugar/phosphate-backbone, is referred to as the DNA-sequence. DNA can be single-stranded or double-stranded. In the double stranded form, the nucleotides of the first strand generally hybridize with the nucleotides of the second strand, eg, A/T-base pairs and G/C-base pairs.

RNA, mRNA: RNA is a general abbreviation for ribonucleic acid. It is a nucleic acid molecule, that is, a polymer made up of nucleotides. These nucleotides are usually adenosine-monophosphate, uridine-monophosphate, guanosine-monophosphate and cytidine-monophosphate monomers linked together along the so-called backbone. The backbone is formed by a phosphodiester linkage between the first sugar proximal to the monomer, namely ribose, and the second phosphate component. A specific chain of monomers is called an RNA-sequence. Ordinarily RNA may be obtainable by transcription of a DNA-sequence, for example inside a cell. In an advancing cell, transcription is usually carried out inside the nucleus or mitochondria. In vivo, transcription of DNA usually results in so-called premature RNA, which must be processed into so-called messenger-RNA, usually abbreviated as mRNA. For example, processing of immature RNA in eukaryotic organisms involves a variety of different post-transcriptional modifications, including splicing, 5' capping, polyadenylation, and export from the nucleus or mitochondria. includes The sum of these processes is called the maturation of the RNA. Mature messenger RNA usually provides a nucleotide sequence that can be translated into the amino acid sequence of a particular peptide or protein. Generally, a mature mRNA comprises a 5' cap, optionally a 5'UTR, an open reading frame, optionally a 3'UTR and a poly(A) sequence. In addition to messenger RNA, several non-coding types of RNA exist, which may be involved in the regulation of transcription and/or translation. The term "RNA" also includes other encoding RNA molecules, such as viral RNA, retroviral RNA and replica RNA.

Sequence of Nucleic Acid Molecules: The sequence of a nucleic acid molecule is generally understood to be a specific and discrete order, ie, a sequence of its nucleotides. The sequence of a protein or peptide is generally understood to be a sequence, ie a sequence of its amino acids.

Sequence homology: Two or more sequences are identical (there are homology) if they exhibit the same length and order of nucleotides or amino acids. Percentage of homology generally describes the extent to which two sequences are identical, ie, it generally describes the percentage of nucleotides whose sequence positions correspond to nucleotides in a reference sequence. For purposes of determining the degree of identity, the sequences being compared are considered to represent the same length, ie, the length of the longest sequence of the sequences being compared. This means that the first sequence of 8 nucleotides is 80% identical to the second sequence of 10 nucleotides comprising the first sequence. In other words, in the present invention, the identity of a sequence preferably relates to the percentage of nucleotides of the sequence having the same position in two or more sequences having the same length. Gaps are usually considered non-identical positions, regardless of their actual positions in alignment.

Stabilized Nucleic Acid Molecules: Stabilized nucleic acid molecules are more susceptible to disintegration or degradation than unmodified nucleic acid molecules, for example by environmental factors or enzymatic digit such as exo- or endonuclease. A more stable, so chemically modified DNA or RNA molecule. In the present invention, a stabilized nucleic acid molecule is stabilized in a cell such as a prokaryotic or eukaryotic cell, a mammalian cell such as a human cell. The stabilizing effect can also be exerted outside the cell, for example, in a buffer solution and the like, for example, in a process for preparing a pharmaceutical composition comprising a stabilized nucleic acid molecule.

Heterologous sequence: In general, two sequences are understood to be 'heterologous' unless they can be derived from the same gene. That is, heterologous sequences may be derived from the same organism, but they do not occur naturally (in nature) in the same nucleic acid as in the same mRNA.

Gene : A gene refers to a specific nucleic acid sequence for producing one or more cellular products and/or for achieving one or more intercellular or intracellular functions. More specifically, the term relates to a nucleic acid section (typically DNA, but RNA in the case of RNA viruses) comprising a nucleic acid encoding a particular protein or functional or structural RNA molecule.

Expression is used in its most general sense and includes the production of RNA or the production of RNA and proteins. This also includes partial expression of nucleic acids. In addition, expression may be transient or stable. In the context of RNA, the term "expression" or "translation" relates to the process by which one strand of messenger RNA directs the assembly of amino acid sequences within the liposome of a cell to make a peptide or protein.

mRNA refers to messenger-RNA and typically relates to a transcript produced using a DNA template and encoding a peptide or protein. Typically, an mRNA comprises a 5'UTR, a protein coding region, a 3'UTR, and a poly(A) sequence. mRNA can be produced by in vitro transcription from a DNA template. In vitro transcription methodologies are well known to those skilled in the art. For example, a variety of in vitro transcription kits are available on the market. According to the present invention, mRNA can also be modified by stabilizing modification and capping.

Open reading frame : may be a sequence of several nucleotide triplets that can generally be translated into peptides or proteins. The open reading frame is preferably a start codon, i.e. a combination of the following three nucleotides (ATG or AUG), usually encoding the amino acid methionine, at its 5' end and a subsequent region usually representing multiple 3 nucleotides in length. contains The ORF is preferably terminated by a stop-codon (eg TAA, TAG, TGA). In general, this is only the stop-codon of the open reading frame. Accordingly, in the present invention the open reading frame preferably begins with a start codon (eg ATG or AUG) and preferably with a stop codon (eg TAA, TGA, or TAG or UAA, UAG, UGA, respectively) ) is a nucleotide sequence consisting of a number of nucleotides that can be divided by three. The open reading frame may be isolated or may be incorporated into a longer nucleic acid sequence, such as a vector or mRNA. The open reading frame may also be referred to as a “protein coding region”.

Bicistronic RNA, multicistronic RNA: Bicistronic or multicistronic RNA typically has two (bicistronic) or more (multicistronic) open reading frames (ORFs). RNA, preferably mRNA. An open reading frame is a sequence of codons that can be translated into a peptide or protein.

G/C chemical modification : The G/C-chemically modified nucleic acid is generally based on a chemically modified wild-type sequence, preferably comprising an increased number of guanosine and/or cytosine nucleotides compared to the wild-type sequence, preferably may be an RNA molecule as defined herein. This increased number can be produced by substitution of codons containing adenosine or thymidine nucleotides with codons containing guanosine or cytosine nucleotides. When abundant G/C content occurs in the coding region of DNA or RNA, this exploits the degeneracy of the genetic code. Thus, codon substitutions preferably do not modify the encoded amino acid residues, but only increase the G/C content of the nucleic acid molecule.

Unmodified reference RNA: RNA that corresponds to a wild-type RNA sequence as it exists in nature. For example, a non-chemically modified reference mRNA corresponds to an mRNA sequence comprising naturally occurring nucleotides and comprises nucleotides comprising a naturally occurring modification, and/or a naturally occurring untranslated region, such as , a naturally occurring 5' cap (CAP) structure m7GpppN, optionally comprising a naturally occurring 5'UTR, if present in the naturally occurring mRNA sequence, and if present in the naturally occurring mRNA sequence and approximately 30 adenosine The wild-type open reading frame, if present in the poly A sequence with

Chimeric Enzyme : Chimeric enzyme refers to an enzyme that is not a natural enzyme found in nature. Thus, a chimeric enzyme may comprise catalytic domains derived from different sources (eg, different enzymes) or catalytic domains derived from the same source (eg, the same enzyme), arranged in a manner different from that found in nature. Chimeric enzymes include monomeric (ie single unit) enzymes as well as oligomeric (ie multiple unit) enzymes, particularly hetero-oligomeric enzymes. Monomeric enzymes refer to single-unit enzymes consisting of only one polypeptide chain.

Immunostimulatory RNA : In the present invention, immunostimulatory RNA (isRNA) may generally be an RNA capable of inducing an innate immune response. It generally does not have an open reading frame and thus does not present a peptide-antigen or immunogen, but elicits an innate immune response, for example, by binding to a specific class of toll-like-receptor (TLR) or other suitable receptor. . But, of course, also mRNAs having an open reading frame and encoding peptides/proteins can induce an innate immune response and thus can be immunostimulatory RNAs.

Immune System : The immune system can protect an organism from infection. If the pathogen successfully penetrates the organism's physical barrier and enters the organism, the innate immune system provides an immediate, but non-specific response. If the pathogen evades this innate response, the vertebrate has a second layer of protection, the adaptive immune system. Here the immune system tailors its response during infection to increase its recognition of the pathogen. This enhanced response, in the form of immunological memory, subsequently remains after the pathogen is eliminated, allowing the adaptive immune system to launch a rapid and powerful attack whenever it is encountered. Accordingly, the immune system includes innate and adaptive immune systems. Each of these two parts contains so-called humoral and cellular components.

Adaptive Immune System: The adaptive immune system is essentially responsible for eliminating or preventing pathogen growth. It generally modulates the adaptive immune response by providing the vertebrate immune system with the ability to recognize and remember specific pathogens (which generate immunity) and launch a strong attack whenever a pathogen is encountered. The system is highly adaptable due to somatic hypermutation (a process of accelerated somatic mutation), and V(D)J recombination (irreversible genetic recombination of antigen receptor gene segments). This mechanism allows for a small number of genes that, after unique expression on each individual lymphocyte, give rise to a vast number of different antigen receptors. Because gene rearrangement causes irreversible modifications to each cell's DNA, its progeny (progeny), including memory B cells and memory T cells, which are key for long-lived specific immunity. offspring)) will then inherit the gene encoding the same receptor specificity.

Innate Immune System: In general, the innate immune system, also known as the non-specific (or non-specific) immune system, includes cells and mechanisms that defend the host from infection by other organisms in a non-specific manner. This means that cells of the innate immune system recognize and respond to pathogens in a normal way, but unlike the adaptive immune system, they do not confer long-lasting or protective immunity to the host. The innate immune system, for example, toll-like receptors (TLRs) or lipopolysaccharidi, TNF-alpha, CD40 ligand, or cytokines, monokini, lymphokines, interleukins ) or chemokines, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 , IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL -25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF , ligands of G-CSF, M-CSF, LT-beta, TNF-alpha, growth factor, and hGH, human toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, Ligand of murine Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13, ligand of NOD-like receptor, RIG-I like receptor It can be activated by ligands, immunostimulatory nucleic acids, immunostimulatory RNA (isRNA), CpG-DNA, antibiotics, or other auxiliary substances such as antiviral agents.

The pharmaceutical composition according to the present invention may comprise one or more such substances. In general, the response of the innate immune system is to recruit immune cells to the site of infection, through the production of chemical factors, including specialized chemical modulators called cytokines; activation of the complement cascade; the selection and removal of foreign substances present in organs, tissues, blood and lymph by specialized white blood cells; activation of the adaptive immune system; and/or acting as physical and chemical barriers to infectious agents.

Immune response : An immune response is usually a specific response of the adaptive immune system (so-called, specific or adaptive immune response) to a specific antigen or a non-specific response of the innate immune system (so-called, non-specific or innate immune response), or a combination thereof. can be

Adaptive Immune Response: The adaptive immune response is generally understood as an antigen-specific response of the immune system. Antigen specificity allows the development of a response tailored to a specific pathogen or pathogen-infected cell. The ability to initiate these tailored responses is maintained in the body by "memory cells". Pathogens have to infect the body more than once, and these specific memory cells are used to quickly clear them. The first step in the adaptive immune response is the activation of naive antigen-specific T cells or different immune cells capable of inducing an antigen-specific immune response by antigen-presenting cells. It occurs within lymphoid tissues and organs through which pure T cells are constantly passing. The three cell types that can serve as antigen presenting cells are dendritic cells, macrophages, and B cells. Each of these cells has a distinct function in inducing an immune response. Dendritic cells can uptake antigens by phagocytosis and micropinocytosis where they differentiate into mature dendritic cells, e.g. by transporting foreign antigens to local lymphoid tissue by contact with them. can be stimulated by Macrophages are induced by infectious agents or other appropriate stimuli that ingest particle antigens such as bacteria and express MHC molecules. The specific ability of B cells to bind and internalize soluble protein antigens via their receptors may also be important for inducing T cells. MHC molecules are normally responsible for presenting antigens to T cells. Presentation of antigens to MHC molecules results in activation of T cells leading to their proliferation and differentiation into armed effector T cells. The most important function of effector T cells is the death of infected cells by CD8+ cytotoxic T cells which together give rise to cell-mediated immunity and the activation of macrophages by Th1 cells, and thus the different types of antibodies, which give rise to a humoral immune response is the activation of B cells by both Th2 and Th1 to produce T cells recognize antigens by their T cell receptors, which do not recognize and bind antigens directly, but instead bind to MHC molecules on the surface of other cells, e.g. pathogen-derived protein antigens, e.g. so-called epitopes ( short peptide fragments of the epitope).

Cellular Immune/Cellular Immune Response: Cellular immunity generally involves the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to antigens. . In a more general manner, cellular immunity is not based on the activation of cells of the immune system as well as antibodies. In general, a cellular immune response is capable of inducing apoptosis, e.g., within a specific immune cell such as a cell that exhibits an epitope of a foreign antigen on their surface, e.g., a dendritic cell or other cell. It can be characterized by the activation of antigen-specific cytotoxic T-lymphocytes. Such cells may be virally infected, infected with intracellular bacteria, or cancer cells displaying tumor antigens. Also, a feature may be the activation of macrophages and natural killer cells capable of destroying pathogens and the stimulation of cells to release various cytokines that affect the functions of other cells involved in the adaptive and innate immune responses.

Immunogen : In the present invention, an immunogen can be generally understood as a compound capable of stimulating an immune response. Preferably, the immunogen is a peptide, polypeptide, or protein. An immunogen of the present invention is a product of translation of a given nucleic acid molecule, preferably a nucleic acid molecule as defined herein. In general, the immunogen elicits at least an adaptive immune response.

Antigen: Antigens are generally capable of being recognized by the immune system and induce an antigen-specific immune response by the adaptive immune system, for example through the formation of antibodies and/or antigen-specific T cells as part of the adaptive immune response. substances that can cause In general, the antigen may be or may comprise a peptide or protein capable of being presented by the MHC to a T cell.

Epitope: Epitopes (also called “antigenic determinants”) can be distinguished into T cell epitopes and B cell epitopes. A portion of a T cell epitope or protein may comprise a fragment having a length of about 6 to about 20 or more amino acids, for example a fragment processed and presented by an MHC class I molecule, from about 8 to about 10 amino acids. , e.g., a fragment having a length of 8, 9, or 10, (or even 11, or 12 amino acids), or a fragment processed and presented by an MHC class II molecule, of about 13 or more amino acids, e.g. for example 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids in length, wherein these segments can be selected from any portion of an amino acid sequence. These fragments are generally recognized by T cells in the form of a complex consisting of a peptide fragment and an MHC molecule, ie the fragments are not generally recognized in their native form. B cell epitopes are generally recognizable by antibodies, i.e., in their native form, having 5 to 15 amino acids, 5 to 12 amino acids, or 6 to 9 amino acids, on the outer surface of a (native) protein or peptide. It is a fragment located in

The epitope of such a protein or peptide may be selected from various variants of such a protein or peptide. Antigenic determinants may be conformational or discontinuous epitopes composed of portions of proteins or peptides, but may be combined with continuous or linear epitopes or three-dimensional structures composed of single polypeptide chains.

Adjuvant/adjuvant component : An adjuvant or adjuvant component in a broad sense is generally a pharmacological and/or immunological agent that can be modified, for example, a drug or other agent such as a vaccine. enhance the effectiveness of This should be interpreted in a broad sense and represents a broad spectrum of substances. In general, these substances are capable of increasing the immunogenicity of an antigen. For example, the adjuvant may be recognized by the innate immune system, eg may elicit the innate immune system. An “adjuvant” generally does not elicit an adaptive immune response. To that extent, an “adjuvant” does not qualify as an antigen. Their mode of action is distinct from the effects induced by antigens as a result of the adaptive immune response.

Protein: In general, a protein comprises one or more peptides or polypeptides. In general, proteins fold into three-dimensional shapes that may be required for proteins to exert biological functions.

Peptide: Generally, a peptide or polypeptide is a polymer of amino acid monomers linked by peptide bonds. It generally contains less than 50 monomer units. However, the term peptide is not a waiver for molecules having more than 50 monomer units. Long peptides, also called polypeptides, generally have 50 to 600 monomer units.

Fragment or part of protein: In the present invention, fragment or part of protein can be generally understood as a peptide corresponding to a continuous part of the amino acid sequence of a protein, preferably from about 6 to about 20 or more amino acids. having a length, e.g., 8, 9, or 10, (or 11), e.g., having a length of e.g. 8, 9, or 10 amino acids , or even 12 amino acids), or fragments processed and presented by MHC class II molecules, preferably about 13 or more amino acids, for example 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids in length, wherein these fragments may be selected from any portion of the amino acid sequence. These fragments can generally be recognized by T cells in the form of a complex consisting of a peptide fragment and an MHC molecule, ie the fragments are generally not recognized in their native form. Fragments or portions of proteins as defined herein may also include epitopes or functional sites of these proteins. A fragment or portion of a protein is an antigen, in particular an immunogen, for example an antigenic determinant (also called an 'epitope') or has antigenic characteristics and elicits an adaptive immune response. Accordingly, fragments of proteins or peptides may comprise at least one epitope of these proteins or peptides. Furthermore, it can be understood that a domain of a protein includes fragments of a protein, such as an extracellular domain, an intracellular domain or a transmembrane domain and short or truncated versions of a protein. .

Transformation : Transformation refers to the introduction of a nucleic acid molecule, DNA or RNA (eg mRNA) molecule into a cell, preferably a eukaryotic cell. In the present invention, transformation includes any method known to a person skilled in the art for introducing a nucleic acid molecule into a cell, a eukaryotic cell such as a mammalian cell. Such methods include, for example, electroporation, for example cationic lipid and/or liposome-based lipofection, calcium phosphate precipitation, nanoparticle-based transformations, virus-based transformations, or transformations based on cationic polymers such as DEAE-dextran or polyethyleneimine. The introduction is non-viral.

Carrier/Polymer Carrier : A carrier may generally be a compound that facilitates transport or complexation of another compound (a cargo). A polymeric carrier is generally a carrier from which a polymer is formed. A carrier may be associated with its cargo by covalent or non-covalent interactions. A carrier is capable of transporting a nucleic acid, eg, RNA or DNA, into a target cell. The carrier may be a cationic component.

Cationic component: The term “cationic component” refers to a molecule that is generally charged, which is generally 1 to 9, less than 9 (eg 5 to 9), or less than 8 (eg 5 to 8), or have a positive charge (cation) of a physiological pH value of less than 7 (eg 5-7), eg 7.3-7.4. Thus, the cationic component may be any positively charged compound or polymer, a cationic peptide or protein that is positively charged under physiological conditions, in particular in vivo physiological conditions. A 'cationic peptide or protein' may contain at least one positively charged amino acid or one or more positively charged amino acids, for example selected from Arg, His, Lys or Orn. Thus, 'polycationic' compounds are also within the range that exhibit more than one positive charge under the given conditions.

Pharmaceutically effective amount : In the present invention, a pharmaceutically effective amount is generally used to replace a pathological level of an expressed peptide or protein, or to replace a missing gene product, e.g., a pathology. It is understood to be an amount sufficient to induce a pharmaceutical effect, such as an immune response, in the case of a hostile situation.

Vehicle: A vehicle is generally understood to be a material suitable for storing, transporting, and/or administering a compound, such as a pharmaceutically active compound. For example, it may be a physiologically acceptable lipid suitable for storage, transport and/or administration of a pharmaceutically active compound.

Vaccine : A vaccine is generally understood to be a prophylactic or therapeutic substance that provides at least one antigen, an immunogen. The antigen or immunogen may be derived from any material suitable for vaccination. For example, the antigen or immunogen may be derived from a pathogen, such as a bacterial or viral particle (particli), or from a tumor or cancerous tissue. An antigen or immunogen stimulates the body's adaptive immune system to provide an adaptive immune response.

Genetic vaccination: Genetic vaccination can generally be understood as vaccination by administration of a nucleic acid molecule encoding an antigen or immunogen or fragment thereof. The nucleic acid molecule may be administered to an individual's body or to an isolated cell of an individual. Upon transformation of specific cells of the body, or upon transformation of isolated cells, antigens or immunogens can be expressed by those cells and subsequently presented to the immune system, eliciting an adaptation, i.e., an antigen-specific immune response. can be Thus, genetic vaccination generally involves a) transformation of a nucleic acid, RNA molecule into a subject, into a patient, or in cells of a patient in vivo/ex vivo or in vitro. administration to isolated cells of a subject, usually a patient; b) transcription and/or translation of the introduced nucleic acid molecule; and optionally c) re-administration of the isolated, transformed cells to the subject, the patient, if the nucleic acid was not directly administered to the patient.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present application fall within the scope of the present application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.

As one of the present invention for achieving the above object,

DdRp RNA expression cassette comprising DNA dependent RNA Polymerase (DdRp) mRNA (capped CM_DdRp mRNA) having a structure capable of intracellular translation and comprising chemically modified nucleotides (CM); and

It provides a self-transcribed RNA/DNA system comprising a; a DNAi expression cassette encoding the mRNA of interest (mRNAi) transcribed by the DdRp, and operatively linked thereto.

As another aspect of the present invention for achieving the above target,

The RNA / DNA system additionally comprises an auto_DdRp expression cassette comprising a promoter capable of binding to DdRp for self-transcription of the DdRp mRNA and DNA encoding DdRp; It provides an RNA/DNA system that does this.

As another aspect of the present invention for achieving the above target,

DdRp RNA expression cassette comprising capped CM_DdRp mRNA; and

For self-transcription of DdRp mRNA, auto_DdRp containing a promoter capable of binding to DdRp and DNA encoding DdRp, and auto_(DdRp + DNAi) expression cassette containing DNAi encoding mRNAi transcribed by the DdRp; and provides a self-transcribed RNA/DNA system characterized in that they are operatively linked. here,

The capped CM_DdRp mRNA, auto_DdRp expression cassette, DNAi expression cassette, or auto_(DdRp+DNAi) expression cassette may include at least one nucleotide sequence modification (SM).

In order to utter the object of the present invention, the DdRp encoded by the bacteriophages T7, T3 and SP6 that is composed of one subunit and does not require other auxiliary elements for enzymatic activity in DdRp of <Table 1> was selected.

DdRp of the auto_DdRp expression cassette of the present invention is DdRp itself, or a catalytic domain of RNA triphosphatase, a catalytic domain of guanylyltransferase, N7-guanine methyltransferase, and It may be a chimeric enzyme comprising a catalytic domain of DNA-dependent RNA polymerase (DdRp), and a method for synthesizing and providing an RNA molecule having a 5'-terminal m7GpppN cap highly translatable by a eukaryotic translation apparatus may be provided.

In the present invention, T7 RNA polymerase (polymerase) is an RNA polymerase that recognizes a phage-specific promoter sequence consisting of about 20 base pairs derived from bacteriophage T7, and catalyzes the synthesis of RNA from DNA in the 5' to 3' end direction. is an enzyme Specifically, unlike eukaryotic RNA polymerase, which requires various intracellular cofactors, T7 RNA polymerase has the advantage of being able to induce transcription without additional intracellular cofactors. In addition, since there is no other RNA polymerase specific for the T7 promoter in the cell of eukaryotes, the transcription system using the T7 promoter/T7 RNA polymerase does not receive interference from other transcription systems in the cell as well as other transcription systems. It does not interfere with the system. On the other hand, other promoters belonging to the same RNA polymerase III type as the T7 promoter, for example, the U1 promoter or the H1 promoter, do not have the property of not causing such interference of the T7 promoter because they already exist in the cell, and the transcription of the present invention The system is not suitable for controlling transcription. More specifically, the T7 RNA polymerase mRNA fragment of the present invention may be composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

In addition, the DdRp RNA expression cassette containing the T7 RNA polymerase mRNA of the present invention may further include a 5'cap structure. The 5'cap structure refers to 7-methylguanosine present at the 5' end of mRNA of most eukaryotic cells and viruses, and mRNA is produced by ribonuclease (RNase). It prevents degradation, and in the cytoplasm, it can bind to a translation initiator and act as a ribosome binding site. In general, the cytoplasmic transcript has a problem in that the translation efficiency by the ribosome is lowered because there is no 5'cap structure essential to the nuclear transcript. Accordingly, the present invention uses a T7 RNA polymerase mRNA fragment having a 5'cap structure to not only increase intracellular stability and intracytoplasmic translation efficiency, but also lower the expression cassette DNA previously used for nuclear-dependent mRNA expression. It is characterized in that it can effectively provide early T7 RNA polymerase in the cytoplasm by overcoming the limitation of nuclear membrane permeation.

In the present invention, in order to synthesize a DdRp RNA expression cassette containing a T7 RNA polymerase mRNA containing a 5′-cap structure and a CM, a dsDNA fragment having a T7 promoter-T7 polymerase sequence is first synthesized by chemical synthesis. (Bioneer), T7 polymerase mRNA having a 5'cap structure and CM-containing T7 promoter-T7 polymerase DNA fragment was synthesized in vitro. T7 polymerase mRNA including the 5'cap structure and CM can exist for a long time by securing in vivo stability.

The DdRp RNA expression cassette encoding the RNA transcribed by DdRp of the present invention comprises a T7 promoter, a DNA encoding the RNA, a poly adenine tail (poly A tail) and a T7 termination sequence, and these are operably linked it could be

In the present invention, by the product of the RNA expression cassette containing the T7 RNA polymerase mRNA, the auto_DdRp DNA expression cassette encoding the T7 RNA polymerase mRNA having the T7 promoter or the DNAi expression cassette having the T7 promoter are also transcribed and amplified. , may be included without limitation in the expression of a specific RNA. In the present invention, T7 RNA polymerase has very high specificity for the T7 promoter, and can transcribe only DNA downstream of the T7 promoter.

Therefore, by using the T7 RNA polymerase in the present invention, it does not react with other promoters in the cell and binds and transcribes the DNA encoding the mRNA having only the T7 promoter, and RNA can be transcribed and amplified with high efficiency. .

Existing expression cassettes, plasmids, or viral systems are nuclear-dependent, requiring transcription using a tool located in the nucleus, so that the existing expression cassette must be transported to the nucleus. However, there is a disadvantage in that less than 1% of the expression cassette introduced through the cell membrane moves to the nucleus.

The expression self-transcription RNA/DNA system of the present invention seeks to overcome the disadvantage of efficiency of transfer to the nucleus by constructing and using a transcription tool in the cytoplasm as shown in <Fig. 3>.

As shown in <Figure 3>, specifically, the DdRp RNA expression cassette is translated by ribosomes in the cytoplasm to generate DdRp, and the generated DdRp recognizes the promoter present in the auto_DdRp expression cassette and transcription of DdRp carry out After that, the generated DdRp mRNA is translated by ribosomes in the cytoplasm again to repeat the production of DdRp, whereby DdRp self-transcription is made. Accordingly, the composition of the present invention is characterized in that the DdRp self-transcribed by the DdRp expression loop can be maintained at a high concentration in the cytoplasm for a long period of time, and mRNAi from the DNAi expression cassette in the cytoplasm by the synthesized DdRp is released for a long period of time. It is characterized by continuous expression and increased expression of the protein of interest.

The present invention provides a DNA comprising a nucleic acid sequence encoding a DdRp RNA expression cassette and a DNAi expression cassette according to the present invention, or both.

Here, the DdRp RNA expression cassette is present in the RNA strand, and the DNAi expression cassette is present in different DNA strands, and the DNAi expression cassette may be transcribed in trans action by DdRp.

Preferably, the DNAi expression cassette capable of being transcribed by the DdRp is at least one selected from DNAi encoding the mRNA of interest.

Here, in addition to the DNA related to the selected mRNA of interest, the DNA encoding the selected specific mRNA of interest artificially synthesized from the outside may be injected into the DNAi expression cassette into the cell.

The present invention provides a DdRp RNA expression cassette according to the present invention, and an auto-DdRp expression cassette comprising a DNA fragment encoding DdRp transcribed by the DdRp in addition to the self-transcribed RNA/DNA system comprising the DNAi expression cassette. can The auto-DdRp expression cassette is capable of executing a DdRp expression loop in which the DdRp DNA fragment downstream of the promoter to which the DdRp binds is repeatedly self-transcribed by the first DdRp synthesized from the DdRp RNA expression cassette.

The present invention provides a DNA comprising a nucleic acid sequence encoding a DdRp RNA expression cassette, an auto-DdRp expression cassette and a DNAi expression cassette, or both, according to the present invention.

In addition, the DdRp included in the auto-DdRp expression cassette may be itself or a DdRp chimeric enzyme having a function of catalyzing the formation of a 5'Cap structure.

In the following, the DdRp RNA expression cassette and the DNAge expression cassette may be separately prepared and co-transfected, and in this case, the auto_(DdRp+DNAge) expression cassette may be used instead.

The present invention provides a method for synthesizing an mRNA and a protein of interest in a cell by a self-transcribed RNA/DNA system, comprising the following steps.

(a) obtaining a DdRp RNA expression cassette;

(b) obtaining a DNAi expression cassette capable of being trans-transcribed by DdRp, and

(c) co-inoculating the DdRp RNA expression cassette and the DNAi expression cassette into the cell;

Here, the DdRp RNA expression cassette includes a 5' cap for inducing translation of DdRp, and additionally, the step of obtaining an auto_DdRp expression cassette may be performed and inoculated together in step (c).

In the method, the DdRp RNA expression cassette and/or the DNAge expression cassette are as defined above for the genome editing RNA/DNA system of the present invention.

The present invention provides a cell comprising a self-transcribed RNA/DNA system. Cells are seeded according to the method according to the invention. Cells are obtainable by the method of the present invention. A cell may be part of an organism.

The present invention provides a method for synthesizing an mRNA and a protein of interest by a self-transcribed RNA/DNA system in a subject, comprising the following steps.

(a) obtaining a DdRp RNA expression cassette;

(b) obtaining a DNAi expression cassette capable of being trans-transcribed by DdRp, and

(c) administering to the subject a self-transcribed RNA/DNA system consisting of a DdRp RNA expression cassette and a DNAi expression cassette;

Here, the DdRp RNA expression cassette includes a 5' cap for inducing translation of DdRp, and additionally, the step of obtaining an auto_DdRp expression cassette may be performed and inoculated together in step (c).

In this method, the DdRp RNA expression cassette and/or the DNAi expression cassette are as defined above for the self-transcribed RNA/DNA system of the present invention.

I. DdRp RNA Expression Cassette

The DdRp RNA expression cassette constituting the self-transcribed RNA/DNA system of the present invention has a structure capable of intracellular translation to increase the expression of DdRp, and CM_DdRp mRNA or capped CM/SM_DdRp mRNA is:

(1) 5'UTR;

(2) mRNA encoding said DdRp; and

(3) 3'UTR;

Nuclear localization signals (NLS) may be located in the DdRp. DdRp without NLS mostly remains in the cytoplasm, whereas protein containing NLS is localized in the nucleus. DdRp without NLS can be cytoplasmic or hybrid DdRp with NLS can be enzymatically activated in the nucleus to selectively start transcription at the T7 promoter placed in chromosomal or plasmid DNA and stop at specific T7 terminator sequences.

Structures that can be translated into cells

CM_DdRp mRNA or CM/SM_DdRp mRNA for expressing DdRp may comprise a 5'UTR comprising a 5'cap or IRES.

The terms "5'cap", "cap", "capped" and ""5'cap structure" are synonymous to refer to a dinucleotide found at the 5' end of some eukaryotic primary transcripts, such as precursor messenger RNA. 5'cap is a structure in which (optionally chemically modified) guanosine is attached to the first nucleotide of an mRNA molecule via 5' in a 5' triphosphate bond (or a chemically modified triphosphate bond in the case of certain cap analogs) The term may refer to a conventional cap or cap analog.

For example, providing RNA with a 5' cap can be accomplished by in vitro transcription of a DNA template in the presence of said 5' cap, wherein said 5' cap is co-transcriptionally incorporated into the resulting RNA strand. Alternatively, the RNA may be generated, for example, by in vitro transcription, and the 5'cap may be attached to the RNA after transcription using a capping enzyme, for example, a capping enzyme of a papillomavirus. In capped RNA, the 3' position of the first base of the (capped) RNA molecule is linked at the 5' position of the subsequent base ("second base") of the RNA molecule via a phosphate diester bond.

The term "conventional 5' cap" means a naturally occurring 5' cap, preferably a 7-methylguanosine cap. In the 7-methylguanosine cap, the guanosine of the cap is a chemically modified guanosine, wherein the modification consists of methylation at the 7-position.

Translation of DdRp is driven by a 5' cap on DdRp mRNA. The 5' cap on DdRp mRNA has a very positive effect on the expression of DdRp as well as the overall performance of the system. Efficient production of trans-encoded target RNA can be achieved.

The SM_DdRp mRNA of the present invention does not contain an internal ribosome entry site (IRES) element. In general, the internal ribosome entry site, abbreviated IRES, is a nucleotide sequence that enables translation initiation from messenger RNA (mRNA) from a location other than the 5' end of the SM_DdRp mRNA sequence, such as in the center of the SM_DdRp mRNA sequence. located in the middle of the sequence.

Substitution of the IRES with the 5' cap in the present invention is a specific modification of the RNA molecule that does not affect the sequence of the polypeptide encoded by the SM_DdRp mRNA molecule (non-polypeptide-sequence modifying modification). For example, in the case of eukaryotic messenger RNA, non-polypeptide-sequence modifying modifications include selection of specific untranslated regions, introduction of introns (eg, rabbit beta-globin intron II sequence), or silent modifications of the coding sequence. , eg, by adaptation to preferential codon usage in a subject cell or subject organism without alteration (silent modification) of the encoded polypeptide sequence.

Production of the target protein encoded by according to the present invention is efficient when the cap is present on the SM_DdRp mRNA.

The presence of a 5' cap in eukaryotic mRNA is thought to play an important role, inter alia, in regulating the intranuclear release of mRNA and facilitating processing, particularly 5' proximal intron excision. The DdRp mRNA of the present invention typically does not need to be exported or processed from the nucleus. Nevertheless, the presence of a 5' cap on the DdRp mRNA is advantageous.

For eukaryotic mRNA, it is generally described that the 5' cap is also usually involved in efficient translation of the mRNA: in general, in eukaryotic cells, translation is initiated only at the 5' end of the messenger RNA (mRNA) molecule unless there is an IRES. .

Eukaryotic cells can present RNA with a 5' cap during intranuclear transcription. Newly synthesized mRNA is usually transformed into a 5' cap structure when the transcript reaches a length of 20 to 30 nucleotides. First, the 5'-terminal nucleotide pppN (ppp represents triphosphate, N represents any nucleoside) is 5' in the cell by a capping enzyme having RNA 5' triphosphatase and guanylyltransferase activity. converted to GpppN. GpppN can then be methylated in the cell by a second enzyme with (guanine-7)-methyltransferase activity to form a mono-methylated m7GpppN cap.

The 5' cap used in the present invention is a natural 5' cap.

In the present invention, natural 5' cap dinucleotides are typically non-methylated cap dinucleotides (G(5')ppp(5')N; also referred to as GpppN) and methylated cap dinucleotides ((m7G(5')ppp) (5')N; also called m7GpppN).

The DdRp mRNA of the present invention does not depend on the capping device of the target cell. When transfected into a subject cell, it is believed that the DdRp mRNA of the present invention is typically not limited to the cell nucleus, ie, the site where capping occurs in a typical eukaryotic cell.

Similar to the IRES in the prior art, the 5' cap on DdRp mRNA is useful for triggering translation initiation of DdRp. The eukaryotic translation initiation factor elF4E may be thought to be involved in the binding of capped mRNAs to ribosomes. The presence of the 5' cap significantly increases performance. Accordingly, the DdRp mRNA of the present invention includes a 5' cap.

The presence of the 5' cap also provides an unexpected synergistic effect in the trans-replication system of the present invention. When the capped DdRp mRNA is provided in trans to RNA, it is more efficient for the capped DdRp mRNA to trigger the production of the protein of interest. Therefore, there is a synergistic effect of 5' cap and replication in trans, that is, an effect superior to replication in cis.

Since the capped RNA of the present invention can be prepared in vitro, it does not depend on the capping device of the target cell. The most frequently used method for forming capped RNAs in vitro is bacteria in the presence of all four ribonucleoside triphosphates and cap dinucleotides such as m7G(5')ppp(5')G (also called m7GpppG). Or to transcribe the DNA template with bacteriophage RNA polymerase.

RNA polymerase then initiates transcription with a nucleophilic attack by the 3'OH of the guanosine moiety of m7GpppG in the α-phosphate of the template nucleoside triphosphate (pppN) to the intermediate m7GpppGpN (where N is the second base of the RNA molecule) is) is formed. Formation of the competing GTP-initiated product pppGpN is inhibited by setting the molar ratio of cap to GTP to 5-10 during in vitro transcription.

A 5' cap is a 5' cap analog. It is particularly suitable when the RNA is obtained by in vitro transcription, for example in vitro transcribed RNA (IVT-RNA). Cap analogs were initially described to facilitate large-scale synthesis of RNA transcripts by in vitro transcription.

In contrast to the engineered replication systems described previously, the DdRp mRNA of the present invention preferably comprises a cap analog. Therefore, the translation of DdRp in the system of the present invention is preferably induced by a cap analog.

Ideally, cap analogs associated with higher translational efficiency and/or increased resistance to degradation in vivo and/or increased resistance to degradation in vitro are selected. Thus, the present invention does not contain any modifications and provides a natural cap [(m7G(5')ppp(5')G; also called m7GpppG, commercially available ScriptCap m7G capping system (which may be added by Epicentre Biotechnology)]] It presents an expression system comprising a.

UTR

An untranslated region (untranslated region) or "UTR" relates to a region that is transcribed but not translated into an amino acid sequence or to the corresponding region in an RNA molecule, such as an mRNA molecule. The UTR may be upstream of the open reading frame and/or downstream of the open reading frame coding for DdRp.

Specifically, in the DdRp mRNA of the present invention, "3'UTR" relates to a region located 3' of the coding region for DdRp, and "5'UTR" refers to a region located 5' of the coding region for DdRp. is about

The 3'UTR, if present, is located at the 3'end of the gene, downstream of the stop codon of the protein coding region, but the "3'UTR" preferably does not include a poly(A) tail. Thus, the 3'UTR is upstream of the poly(A) tail, eg directly adjacent to the poly(A) tail (if present).

The 5'UTR, if present, is located at the 5' end of the gene upstream of the start codon of the protein coding region. The 5'UTR is downstream of the 5' cap, eg immediately next to the 5' cap (if present).

The 5' and/or 3'UTR may be functionally linked with the open reading frame, such that these regions are associated with the open reading frame to increase stability and/or translation efficiency of RNA comprising the open reading frame.

UTR is involved in RNA stability and translation efficiency, which are prerequisites for effective transport using RNA transport. By selecting specific 5' and/or 3' untranslated regions (UTRs), both can be improved, in addition to structural modifications regarding the 5' cap and/or 3' poly(A)-tail.

Sequence elements within the UTR are generally understood to affect translation efficiency (mainly 5'UTR) and RNA stability (mainly 3'UTR). It is preferred that an activated 5'UTR is present in order to increase the translation efficiency and/or stability of the nucleic acid sequence. Independently or additionally, it is preferred that an activated 3'UTR is present to increase the translation efficiency and/or stability of the nucleic acid sequence.

Preferably, the DdRp mRNA comprises a 5'UTR and/or a 3'UTR that is not heterologous or native to that from which the DdRp is derived. This allows the design of untranslated regions according to the desired translation efficiency and RNA stability.

The 3'UTRs of the present invention have one or more foldables to provide a stem-loop structure that interacts with proteins (such as RNA-binding proteins) known to act as barriers for exoribonucleases or to increase RNA stability. Reverse iterations may be included.

Two consecutive identical copies of human beta-globin 3'UTR, in particular human beta-globin 3'UTR, contribute to high transcriptional stability and translational efficiency.

Thus, the DdRp mRNA of the present invention comprises two consecutive identical copies of human beta-globin 3'UTR. Thus, in the 5'→3' direction (a) optionally 5'UTR; (b) an open reading frame encoding DdRp; (c) 3'UTR; 3'UTR comprising two consecutive identical copies of human beta-globin 3'UTR, a fragment thereof, or a variant or fragment thereof of human beta-globin 3'UTR.

The DdRp mRNA of the present invention activates to increase the translation efficiency and/or stability of but not human beta-globin 3'UTR, a fragment thereof, or a variant of human beta-globin 3'UTR or a fragment thereof, 3'UTR include

The DdRp mRNA of the present invention contains an activated 5'UTR to increase translation efficiency and/or stability.

The UTR-containing RNA according to the invention can be prepared, for example, by in vitro transcription. These can be achieved by genetically modifying the expression of a nucleic acid molecule (eg DNA) of the invention in a manner that allows transcription of RNA with 5'UTR and/or 3'UTR.

A 3'UTR is a portion of an mRNA that is usually located between the protein coding region (ie, open reading frame) of the mRNA and the poly(A) sequence. The 3'UTR of the mRNA is not translated into the amino acid sequence. The 3'UTR is usually encoded by a gene that is transcribed into the respective mRNA during the gene expression process. The genomic sequence is first transcribed into immature mRNA containing selective introns. Thereafter, the immature mRNA is further processed to undergo a maturation process to become a mature mRNA. This maturation process includes 5' capping, splicing of immature mRNA to cut additional intro, and polymerization of adenylate at the 3' end of immature mRNA and additional endo- or exo nucleases. Modifications at the 3' end, such as cleavage, etc. are included. In the present invention, the 3'UTR corresponds to the sequence of a mature mRNA located at the 3' end of the stop codon of the protein coding region, preferably immediately adjacent to the 3' end of the stop codon of the protein coding region, and the poly(A) sequence It extends to the 5' side of the, preferably the nucleotide immediately adjacent to the 5' end of the poly(A) sequence. In the present invention, the 3'UTR of a gene, such as the 3'UTR of alpha or beta globin, is a sequence corresponding to the 3'UTR of a mature mRNA derived from this gene, i.e., obtained by transcription of the gene and maturation of immature mRNA. sequence corresponding to mRNA. The term "3'UTR of a gene" includes DNA sequences and RNA sequences of 3'UTRs.

A 5'UTR is generally understood as a specific section of messenger RNA (mRNA). It is located 5' of the open reading frame of the mRNA. Generally, the 5'UTR starts with a transcriptional start site and ends one nucleotide before the start codon of the open reading frame. The 5'UTR may include elements for regulating gene expression, also called regulatory elements. Such a regulatory element may be, for example, a ribosome binding site or a 5'-terminal oligopyrimidine tract. The 5'UTR can be modified post-transcriptionally, for example, by the addition of a 5' cap. In the present invention, the 5'UTR corresponds to the sequence of the mature mRNA located between the 5'cap and the start codon. Preferably, the 5'UTR is from the nucleotide located 3' of the 5' cap, preferably from the nucleotide located immediately 3' of the 5' cap, to 5' of the start codon of the protein coding region. to the nucleotide located, preferably to the nucleotide located immediately 5' of the start codon of the protein coding region. The nucleotide located immediately 3' of the 5' cap of the mature mRNA generally corresponds to the start of transcription.

The 5'UTR sequence may be an RNA sequence, such as an mRNA sequence used to define a 5'UTR sequence, or a DNA sequence corresponding to such an RNA sequence. The 5'UTR of a gene is a sequence corresponding to the 5'UTR of a mature mRNA derived from this gene, ie, a sequence corresponding to an mRNA obtained by transcription of the gene and maturation of an immature mRNA. The 5'UTR of a gene includes the DNA sequence and the RNA sequence of the 5'UTR.

The 5' terminal oligopyrimidine tube (Oligopyrimidine Tract, TOP) is generally a 5' terminal region of a nucleic acid molecule, such as a 5' terminal region of a specific mRNA molecule or a functional entity of a specific gene, for example, the 5' end of a transcription region. A stretch of pyrimidine nucleotides located in the region. The sequence usually begins with a cytidine corresponding to the start of transcription, usually followed by a stretch of about 3 to 30 pyrimidine nucleotides. Pyrimidine Stretch The 5' TOP is thus terminated with one nucleotide 5' of the first purine nucleotide located downstream of the TOP. A messenger RNA containing a 5'-terminal oligopyrimidine tube is often referred to as TOP mRNA. Thus, a gene providing such messenger RNA is referred to as a TOP gene. TOP sequences are found, for example, in mRNA and ribosomal proteins encoding genes and peptide elongation factors.

In the present invention, a TOP motif is a nucleic acid sequence corresponding to 5'TOP as defined above. Therefore, in the present invention, the TOP motif is preferably a stretch of pyrimidine nucleotides having a length of 3 to 30 nucleotides. Preferably, the TOP-motif is at least 3 pyrimidine nucleotides, preferably at least 4 pyrimidine nucleotides, preferably at least 5 pyrimidine nucleotides, more preferably at least 6 nucleotides, even more preferably at least 7 It consists of nucleotides, most preferably at least 8 pyrimidine nucleotides, wherein the stretch of pyrimidine nucleotides preferably starts at its 5' end with a cytosine nucleotide. In TOP genes and TOP mRNAs, the TOP-motif preferably starts at its 5' end with the start of transcription and ends with one nucleotide 5' of the first purine residue in said gene or mRNA. In the sense of the present invention, the TOP motif is preferably located at the 5' end of the sequence representing the 5'UTR or at the 5' end of the sequence encoding the 5'UTR. Thus, preferably, if this stretch is derived from the 5' end of the respective sequence, such as the 5'UTR element of the SM_mRNAi according to the invention, the 5'UTR element of the SM_mRNAi according to the invention, or the 5'UTR of the TOP gene as described herein. A stretch of three or more pyrimidine nucleotides is called a "TOP motif" in the sense of the present invention if it is located in a nucleic acid sequence. In other words, a stretch of 3 or more pyrimidine nucleotides that are not located at the 5' end of the 5'UTR or 5'UTR element and are located anywhere within the 5'UTR or 5'UTR element is preferably not referred to as a "TOP motif" .

TOP genes are generally characterized by the presence of a 5' terminal oligopyrimidine tube. Moreover, most TOP genes are characterized by growth-associated translational regulation. However, TOP genes with tissue-specific translational regulation are also known. The 5'UTR of the TOP gene corresponds to the sequence of the 5'UTR of the mature mRNA derived from the TOP gene, and extends from the nucleotide located 3' of the 5'cap (CAP) to the nucleotide located 5' of the start codon. The 5'UTR of a TOP gene generally does not contain any start codons, upstream AUGs (uAUGs) or upstream open reading frames (uORFs). Therein, upstream AUGs and upstream open reading frames are generally understood as AUGs and open reading frames that appear 5' of the start codon (AUG) of the open reading frame to be translated. The 5'UTR of TOP genes is generally relatively short. The 5'UTR length of a TOP gene can vary from 20 nucleotides to 500 nucleotides, and is generally less than about 200 nucleotides, less than about 150 nucleotides, and less than about 100 nucleotides. In the sense of the present invention, an example of a 5'UTR of a TOP gene is a nucleic acid sequence extending from the nucleotide at position 5 to the nucleotide located immediately 5' of the start codon (eg, ATG). A particularly preferred fragment of the 5'UTR of the TOP gene is the 5'UTR of the TOP gene lacking the 5'TOP motif. The '5'UTR of the TOP gene' preferably refers to the 5'UTR of the naturally occurring TOP gene.

poly(A) sequence

The DdRp mRNA and mDdRp RNA expression cassettes of the present invention comprise a 3' poly(A) sequence.

The poly(A) sequence is essentially at least 20, at least 26, at least 40, at least 80, at least 100, no more than 500, no more than 400, no more than 300, no more than 200, in particular no more than 150 A nucleotides, in particular consists of or comprises about 120 A nucleotides.

Depending on the number of nucleotides in the poly(A) sequence, typically at least 50%, at least 75% of the majority of the nucleotides in the poly(A) sequence are A nucleotides, but the remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (free delay). ), G nucleotides (guanylate), C nucleotides (cytidylates). All nucleotides in the poly(A) sequence, i.e. 100% according to the number of nucleotides in the poly(A) sequence, are A nucleotides. The term “A nucleotide” or “A” refers to adenylate.

The present invention is based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand, a 3' poly(A) attached during RNA transcription, ie during the production of transcribed RNA in vitro. ) provides the sequence. The DNA sequence (coding strand) encoding the poly(A) sequence is referred to as the poly(A) cassette.

The 3' poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides, but is interrupted by a random sequence with an even distribution of 4 nucleotides (dA, dC, dG, dT). Such random sequences may be 5-50 in length, preferably 10-30, more preferably 10-20.

A poly(A) cassette consisting essentially of dA nucleotides, but having an even distribution of 4 nucleotides (dA, dC, dG, dT) and interrupted by a random sequence of e.g. 5-50 nucleotides in length, is a DNA It exhibits constant proliferation of plasmid DNA in E. coli at the RNA level and is still associated with beneficial properties with respect to supporting RNA stability and translation efficiency at the RNA level.

As a result, the 3' poly(A) sequence contained in the RNA molecules described herein consists essentially of A nucleotides, but by a random sequence with an even distribution of 4 nucleotides (A, C, G, U). is stopped Such random sequences may be 5-50, 10-30, 10-20 in length.

Polyadenylation is generally understood as the addition of a poly(A) sequence to a nucleic acid molecule such as an RNA molecule, eg, immature mRNA. The adenylic acid polymerization reaction can be induced by a so-called adenylic acid polymerization reaction signal. This signal is located within a stretch of nucleotides at the 3' end of a nucleic acid molecule, such as an RNA molecule, to be subjected to adenylic acid polymerization. The adenylic acid polymerization signal generally includes a hexamer consisting of adenine and uracil/thymine nucleotides, and the hexamer sequence AAUAAA. Other sequences, hexamer sequences, are possible. Adenylic acid polymerization occurs during processing of pre-mRNA (also called immature-mRNA). RNA maturation (from pre-mRNA to mature mRNA) involves the step of adenylate polymerization.

Codon usage

In general, the degeneracy of the genetic code makes it possible to replace certain codons (base triplets encoding amino acids) present in RNA sequences by other codons (base triplets) while maintaining the same coding capacity (replacement of a codon is encodes the same amino acid as the codon replaced).

In the present invention, at least one codon of the open reading frame included in the RNA molecule is different from each codon of each open reading frame in the species from which the open reading frame is derived. A coding sequence in an open reading frame is said to be "altered".

For example, when the coding sequence of the open reading frame is modified, frequently used codons may be selected. Modifications in the coding sequence of nucleic acid molecules, including substitution of more frequently used rare codons.

Because the frequency of codon usage varies depending on the subject cell or subject organism, this type of modification is appropriate to tailor the nucleic acid sequence to expression in a particular subject cell or subject organism. Generally speaking, more frequently used codons are typically translated more efficiently in a subject cell or subject organism, but modification of all codons in the open reading frame is not always necessary.

For example, if the coding sequence of the open reading frame is modified, the content of G (guanylate) residues and C (cytdylate) residues can be modified by selecting the codon with the most abundant GC-content for each amino acid. can It has been reported that RNA molecules with GC-rich open reading frames have the potential to decrease immune activity and enhance translation and half-life of RNA.

Each of the open reading frames coding for DdRp according to the present invention can be adapted.

II. DNA expression cassette

The self-transcribed RNA/DNA system of the present invention may include a DNAi expression cassette encoding mRNAi and an auto_DdRp expression cassette encoding DdRp or an auto_(DdRp+DNAi) expression cassette encoding mRNAi and DdRp.

The DdRp DNA expression cassette for the purpose of preparing the DdRp RNA expression cassette of the present invention is

(1) a promoter to which DdRp binds;

(2) 5'UTRs;

(3) a DNA fragment encoding the DdRp; and

(4) 3' UTR;

A DdRp DNA expression cassette for the purpose of preparing the DdRp RNA expression cassette may be used as an auto_DdRp expression cassette.

Preferably, the DNAi expression cassette of the present invention comprises

(1) a promoter to which DdRp binds;

(2) 5'UTRs;

(3) a DNA fragment encoded by mRNAi; and

(4) 3' UTRs; and may be configured to be operatively linked.

Preferably, the auto_(DdRp+DNAi) expression cassette of the present invention is

(1) a promoter to which DdRp binds;

(2) 5'UTRs;

(3) DNA fragments encoded by the DdRp and mRNAi; and

(4) 3'UTR; comprising, wherein they are operatively linked;

Here, the DNA encoding DdRp and mRNAi may have a promoter to which DdRp binds in each upstream direction, or a promoter to which one DdRp binds in an upstream direction.

For DdRp of the present invention, DdRp composed of one subunit was selected.

The DdRp of the present invention increases the expression of any one selected from the group comprising T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase in the cytoplasm in a nuclear-independent manner, thereby continuously expressing target RNA for a long time. there is.

In the present invention, T7 RNA polymerase has very high specificity for the T7 promoter, and can transcribe only DNA downstream of the T7 promoter. Therefore, by using T7 RNA polymerase in the present invention, binding and transcription to the DdRp RNA expression cassette and auto_DdRp expression cassette having only the T7 promoter without reacting with other promoters in the cell are performed, and T7 RNA polymerase itself with high efficiency can be amplified by self-transcription and transcription of RNA.

The auto_DdRp expression cassette is characterized in that the T7 RNA polymerase is self-transcribed in the cytoplasm by repetition of the T7 RNA polymerase expression loop.

Specifically, the auto_DdRp expression cassette includes a T7 promoter, an internal ribosome entry site (IRES) region, a gene encoding T7 RNA polymerase, a poly A tail, and a T7 termination sequence, and they operate It may be configured in a possible connection. The IRES region included in the auto_DdRp expression cassette may be a viral IRES, and by generating an IRES-fused T7 polymerase transcript, not only increases the transcriptional stability of DdRp mRNA, but also increases the expression efficiency of T7 polymerase in the cytoplasm. can keep

The self-transcription may be performed by the DdRp expression loop by the auto_DdRp expression cassette for self-transcription of DdRp mRNA by the DdRp RNA expression cassette constituting the composition of the present invention.

Specifically, as shown in <Fig. 3>, the DdRp RNA expression cassette is translated by ribosomes in the cytoplasm to generate DdRp, and the generated DdRp recognizes a promoter present in the auto_DdRp expression cassette and transcription of DdRp mRNA carry out After that, the generated DdRp mRNA is translated by ribosomes in the cytoplasm again to repeat the production of DdRp, whereby self-transcription of DdRp mRNA is made. Therefore, the composition of the present invention is characterized in that it can maintain the DdRp mRNA self-transcribed by the DdRp expression loop at a high concentration for a long time in the cytoplasm.

In the present invention, the cytoplasmic expression of T7 polymerase over time in cells treated with the DOPC liposome delivery system (auto_mRNAi@LS) containing the auto_DdRp expression cassette was examined by Western blot. It was confirmed that the above-mentioned high concentration was maintained.

As shown in <Figure 3>, the present invention provides a DdRp RNA expression cassette; And DdRp is maintained at a high concentration in the cytoplasm for a long period of time through a DdRp expression loop carried by an auto_DdRp expression cassette comprising a DNA encoding DdRp for DdRp self-transcription, and thus mRNAi is also continuously expressed for a long time. .

Capping is a special structure found at the 5' end of almost all eukaryotic messenger RNAs. The simplest cap structure, cap0, is the result of guanine nucleoside addition methylated at N7 bound by a gamma phosphate at the 5'5' triphosphate end bound to the end of the primary RNA (i.e., m ⁷ GpppN, where N is any base, p represents phosphate and m is a methyl group). The formation of cap0 involves a series of three enzymatic reactions. RNA triphosphatase (RTPase) removes the gamma phosphate residue at the 5' triphosphate terminus of the diphosphate-generating pre-mRNA, and RNA guanylyltransferase (GTase) transfers GMP from GTP to the diphosphate-generating RNA terminus. and RNA N7-guanine methyltransferase (N7-MTase) adds a methyl residue on nitrogen 7 of guanine to the GpppN cap.

In more advanced eukaryotes and some viruses, primary (ie, cap1 construct; m ⁷ GpppNm ^2'-o pN) and secondary (ie, cap2 externa; m ⁷ GpppNm ^2'-o pNm ^2'-o ) transcription The 2'-hydroxyl group of the ribose of a given nucleotide can be methylated by two distinct ribose-2'-O MTases, termed cap1- and cap2-specific MTases, respectively.

However, in contrast to cellular N7-MTase activity, which is only present in the nucleus, cap1 ribose-2'-O MTase activity was detected both in the cytoplasm and nucleus of HeLa cells, whereas cap2 MTase activity was found only in their cytoplasm. became

Formation of the 5'-end m ⁷ GpppN cap is the first step in pre-mRNA processing. The m ⁷ GpppN cap plays an important role in mRNA stability and its transport from the nucleus to the cytoplasm.

Furthermore, the 5'-terminal m ⁷ GpppN cap is important for the translation of mRNA into protein by anchoring the eukaryotic translation initiation factor 4F (eIF4F) complex, which mediates the recruitment of the 16S portion of the small ribosomal subunit into mRNA. . Thus, the 5'-terminal m ⁷ GpppN cap significantly enhances the translation of mRNA both in vitro and in vivo.

DdRp of the auto_DdRp expression cassette of the present invention is DdRp itself, or a catalytic domain of RNA triphosphatase, a catalytic domain of guanylyltransferase, N7-guanine methyltransferase, and It is reported that a chimeric enzyme comprising a catalytic domain of DNA-dependent RNA polymerase (DdRp) synthesized an RNA molecule having a 5'-terminal m7GpppN cap highly translatable by a eukaryotic translation apparatus without induction of cytotoxicity and apoptosis ( It can be prepared using a chimeric enzyme based on Korea, Patent Publication No. 10-2013-0069632).

The present invention relates to at least one, in particular catalytic domain of RNA triphosphatase, at least one, in particular catalytic domain of guanylyltransferase, at least one, in particular catalytic domain of N7-guanine methyltransferase, and at least one, in particular It relates to a chimeric enzyme comprising the catalytic domain of DdRp.

In particular, the chimeric enzyme according to the present invention is capable of synthesizing RNA molecules having a 5'-terminal m7 GpppN cap.

A chimeric enzyme refers to an enzyme that is not a natural enzyme found in nature. Thus, a chimeric enzyme may comprise catalytic domains derived from different sources (eg, different enzymes) or catalytic domains derived from the same source (eg, the same enzyme), arranged in a manner different from that found in nature. Chimeric enzymes include monomeric (ie single unit) enzymes as well as oligomeric (ie multiple unit) enzymes, particularly hetero-oligomeric enzymes. Monomeric enzymes refer to single-unit enzymes consisting of only one polypeptide chain.

In particular, the catalytic domains of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase and DdRp are covalently and/or non-covalently linked together.

In particular, the catalytic domains of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase and DdRp are functionally linked together to synthesize an RNA molecule with a 5'-terminal m7 GpppN cap.

In particular, at least one, in particular a catalytic domain of RNA triphosphatase, at least one, in particular catalytic domain of guanylyltransferase, at least one, in particular catalytic domain of N7-guanine methyltransferase and at least one, in particular DdRp catalytic domains, wherein at least two of said catalytic domains are linked together covalently or non-covalently, preferably at their termini (N or C terminus), more particularly said at least one, in particular catalytic domain of RNA triphosphatase , said at least one, in particular catalytic domain of guanylyltransferase, at least one, in particular at least one catalytic domain selected from the group consisting of catalytic domain of N7-guanine methyltransferase, preferably at its terminus (N or C The chimeric enzyme of the present invention comprising a covalently or non-covalently linked (terminus)) is linked with said at least one, in particular, the catalytic domain of DdRp, preferably at its terminus (N or C terminus).

In particular, the present invention relates to at least one, in particular a catalytic domain of RNA triphosphatase, at least one, in particular a catalytic domain of guanylyltransferase, at least one, in particular a catalytic domain of N7-guanine methyltransferase, and at least one, In particular, it relates to the chimeric enzyme of the present invention comprising the catalytic domain of DdRp, provided that the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase, the catalytic domain of N7-guanine methyltransferase and the nuclear eukaryotic DdRp I , the only catalytic domain of II and/or III; and more particularly, chimeric enzymes comprising the only catalytic domain(s) of DdRp II.

In particular, the present invention relates to at least one, in particular a catalytic domain of RNA triphosphatase, at least one, in particular a catalytic domain of guanylyltransferase, at least one, in particular a catalytic domain of N7-guanine methyltransferase, and at least one, In particular, it relates to the chimeric enzyme of the present invention comprising the catalytic domain of DdRp, provided that the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase, the catalytic domain of N7-guanine methyltransferase and the nuclear eukaryotic DdRp I , a catalytic domain of II and/or III; and more particularly, chimeric enzymes comprising at least one catalytic domain of DdRp II.

In particular, when expressed in a eukaryotic host cell, the chimeric enzyme according to the present invention is capable of synthesizing an RNA molecule having a 5' terminal m7 GpppN cap, preferably translatable by a eukaryotic translation apparatus.

In particular, when expressed in eukaryotic host cells, the catalytic domains of RNA triphosphatase, guanylyltransferase and N7-guanine methyltransferase bind the m7 GpppN cap at the 5' end of the RNA molecule synthesized by the catalytic domain of DdRp. The RNA molecule, which can be added, preferably having a 5'-terminal m7 GpppN cap, can be translated by a eukaryotic translation apparatus.

In particular, when expressed in eukaryotic host cells, when the catalytic domain of DdRp is the catalytic domain of bacteriophage DdRp, the catalytic domains of RNA triphosphatase, guanylyltransferase and N7-guanine methyltransferase are at their 5' ends m7GpppN cap can be added to the 5' end of the RNA molecule having a guanosine ribonucleotide in

In particular, when expressed in a eukaryotic host cell, when the catalytic domain of DdRp is the catalytic domain of bacterial DdRp, the catalytic domains of RNA triphosphatase, guanylyltransferase and N7-guanine methyltransferase are At the 5' end of the RNA molecule having guanosine or adenosine ribonucleotides, an m7GpppN cap can be added.

In particular, when expressed in eukaryotic host cells, when the catalytic domain of DdRp is the catalytic domain of human or mouse mitochondrial DdRp, the catalytic domains of RNA triphosphatase, guanylyltransferase and N7-guanine methyltransferase are An m7GpppN cap can be added at the 5' end of the RNA molecule with adenosine or thymidine ribonucleotides at the 5' end.

The catalytic domain of an enzyme relates to a domain that is necessary and sufficient to ensure enzyme function, especially in its three-dimensional structure. For example, the catalytic domain of RNA triphosphatase is a domain necessary and sufficient to ensure RNA triphosphatase function. The term “catalytic domain” includes the catalytic domain of a wild-type or mutant enzyme.

The chimeric enzyme of the present invention includes at least the catalytic domain, but may further include all or a part of the enzyme containing the catalytic domain. Indeed, according to one aspect of the chimeric enzyme according to the invention, said catalytic domain of DdRp may be comprised in all or part of a DdRp, preferably a monomeric DdRp. Said catalytic domain of RNA triphosphatase, said catalytic domain of guanylyltransferase and said catalytic domain of N7-guanine methyltransferase may also be included in all or part of a capping enzyme, preferably a monomeric capping enzyme.

The chimeric enzyme of the present invention may be a nuclear enzyme, a subcellular compartment enzyme or a cytoplasmic enzyme. Thus, the chimeric enzyme according to the present invention may comprise a signal peptide or marker-signal known to one of ordinary skill in the art that induces delivery of the enzyme in a cell. For example, the chimeric enzyme according to the present invention may include a nuclear localization signal (NLS) that directs the enzyme to the nucleus. These NLSs are often units of five basic positively charged amino acids. The NLS can be located anywhere on the peptide chain.

A chimeric enzyme does not contain a signal peptide or marker-signal that induces the transfer of the enzyme, except to the cytoplasm, when it is a cytoplasmic chimeric enzyme.

Cytoplasmic localization of the chimeric enzyme of the present invention optimizes transgene expression levels by avoiding the vigorous movement of large DNA molecules (i.e., transgenes) from the cytoplasm to the nucleus of eukaryotic cells and export of RNA molecules from the nucleus to the cytoplasm. have an advantage

Accordingly, these cytoplasmic chimeric enzymes of the present invention may be useful for generating host-independent, eukaryotic gene expression systems capable of operating in the cytoplasm where significantly higher amounts of transfected DNA are normally found compared to the nucleus.

These cytoplasmic chimeric enzymes of the present invention are capable of synthesizing RNA molecules with a 5'-terminal m7GpppN cap, highly translatable by the eukaryotic cytoplasmic translation apparatus, without cytotoxicity and induction of apoptosis.

Since endogenous gene transcription occurs in the nucleus of eukaryotic cells, as opposed to transgene transcription that occurs in the cytoplasm, there is no competition between endogenous gene residues and transgene transcription.

Therefore, the cytoplasmic chimeric enzyme of the present invention is particularly suitable for large-scale analysis and protein production.

In the chimeric enzyme of the present invention, the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase and the catalytic domain of N7-guanine methyltransferase are contained in a monomer, ie, one polypeptide. For example, the monomer may be a monomeric capping enzyme or a monomeric chimeric enzyme according to the present invention.

In particular, the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase and the catalytic domain of N7-guanine methyltransferase are comprised in a monomeric capping enzyme. In this case, the chimeric enzyme of the present invention comprises a monomeric capping enzyme, wherein the enzyme comprises the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase and the catalytic domain of N7-guanine methyltransferase. do. The monomeric capping enzyme is, in particular, a wild-type Bluetooth virus (BTV) capping enzyme, a wild-type Brome mosaic virus (BMV) capping enzyme, and a wild-type African swine virus capable of adding an m7GpppN cap to the 5' end of the RNA molecule. A monomeric virus capping enzyme selected from the group consisting of fever virus (ASFV) capping enzymes, wild-type acanthamoeba polyphaga mimivirus capping enzymes and mutants and derivatives thereof, and more particularly wild-type African swine fever virus It may be a capping enzyme.

In particular, said catalytic domain of DdRp may also be comprised in a monomer, ie in one polypeptide.

For example, the monomer may be a monomeric DdRp or a monomeric chimeric enzyme according to the present invention.

In particular, said catalytic domain of DdRp is comprised in monomeric DdRp. In this case, the chimeric enzyme of the present invention contains a monomeric DdRp comprising the catalytic domain of DdRp. The monomeric DdRp may be a monomeric phage DdRp, in particular T7 RNA polymerase, T3 RNA polymerase and SP6, capable of synthesizing single-stranded RNA having sequence complementarity to double-stranded template DNA in the 5' to 3' direction. T7 RNA polymerase, which may be selected from the group consisting of RNA polymerase and mutants or derivatives thereof, and more particularly, capable of synthesizing single-stranded RNA having sequence complementarity to double-stranded template DNA in the 5' to 3' direction; and It may be selected from the group consisting of mutants or derivatives thereof.

At least two selected from the group consisting of the catalytic domain of DdRp and the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase and the catalytic domain of N7-guanine methyltransferase, and more preferably the entire catalytic domain is may be included in the monomer.

The chimeric enzymes of the present invention may be monomeric or oligomeric. Indeed, the catalytic domains of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase and DdRp may be included in one or several polypeptides.

Preferably, the chimeric enzyme of the present invention may be monomeric.

Indeed, the present invention relates to a monomeric chimeric enzyme comprising the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase, the catalytic domain of N7-guanine methyltransferase and the catalytic domain of DdRp, without inducing cytotoxicity and apoptosis. , can synthesize RNA molecules with a 5' terminal m7GpppN cap, which are highly translatable by eukaryotic translation machinery.

It is not self-evident that capping of transcripts occurs with monomeric enzymes, due to steric hindrances and components and enzymes that must remain in their native structure. Indeed, capping of the T7 transcript cannot be achieved by the fusion enzyme CTD-T7 RNA polymerase, even assuming that it causes an m7GpppN cap at the 5' end of the developing RNA molecule.

The monomeric chimeric enzymes of the present invention have the advantage of being particularly inexpensive, rapid and easy to implement, making them highly suitable for large-scale assays and protein production. Indeed, the production of monomeric enzymes is easier than oligomeric enzymes. Also, since only single-units exist, there is no problem of unit assembly. Monomeric enzymes are also easier to manipulate than multimeric enzymes.

DdRp (RNAP) relates to a nucleotidyl transferase that synthesizes RNA with sequence complementarity to double-stranded template DNA in the 5'→3' direction.

Preferably, said catalytic domain of DdRp is the catalytic domain of an enzyme, which has a relatively simple structure and more preferably characterizes genomic enzyme regulatory elements (ie promoter, transcription termination signal and chain splicing). Thus, in particular, the catalytic domain of DdRp may be the catalytic domain of a bacteriophage DdRp, a bacterial DdRp or a DdRp of various eukaryotic organelles (eg, mitochondria, chloroplasts and prochromosomes).

In the present invention, the catalytic domain of DdRp is the catalytic domain of bacteriophage DdRp. Bacteriophage DdRp has a significant advantage in optimizing transgene expression levels, especially by having a higher processing capacity than eukaryotic RNA polymerase. Bacteriophage DdRp also has a much simpler structure than most nuclear eukaryotic polymerases, which have a complex structure with multiple subunits (eg RNA polymerase II). Most bacteriophage DdRp characterized to date are single-subunit enzymes, which do not require helper proteins for initiation, elongation or termination of transcription. Several of these enzymes named for cloned bacteriophages also have well-characterized regulatory genomic elements (ie, promoters, termination signals, transcriptional stop sequences), which are important for transduction.

Also, there is no competition between endogenous gene transcription and transgene transcription. Chimeric enzymes according to the invention comprising bacteriophage DNA dependent RNA-polymerase residues enable the production of RNA transcripts in any eukaryotic species (eg yeast, rodent and human). They are inexpensive, rapid, and easy to implement, making them suitable for large-scale analysis and protein production, and in contrast to eukaryotic RNA polymerases such as RNA polymerase II, most bacteriophage DdRp do not contain relevant factors for initiation, elongation or termination of transcription. Because it is not required, it enables the production of RNA transcripts in any biological system (eg, cellular reaction mixtures, cultured cells and living organisms).

Said catalytic domain of bacteriophage DdRp is the catalytic domain of bacteriophage DdRp, in particular wild type of T7 RNA polymerase, wild type of T3 RNA polymerase (NCBI genomic sequence ID# NC_003298; GeneID# 927437; UniProtKB/Swiss-Prot ID# Q778M8), K11 Wild type of RNA polymerase (NCBI genomic K11 RNAP sequence ID# NC_004665; GeneID# 1258850; UniProtKB/Swiss-Prot ID# Q859H5), wild type of ψA1122 RNA polymerase (NCBI genome sequence ID# NC_004777; GeneID# 1733944; UniProtKB/Swiss) -Prot protein ID# Q858N4), wild-type of ψYeo3-12 RNA polymerase (NCBI genomic sequence ID# NC_001271; GeneID# 1262422; UniProtKB/Swiss-Prot ID# Q9T145) and wild-type of gh-1 RNA polymerase (NCBI genomic sequence) ID# NC 004665; GeneID# 1258850; UniProtKB/Swiss-Prot protein ID#Q859H5), wild type of K1-5 RNAP RNA polymerase (NCBI genome sequence ID# NC_008125; GeneID# 5075932 UniProtKB/Swiss-Prot protein ID# Q8SCG8) and wild-type SP6 RNA polymerase (NCBI genome sequence ID# NC_004831; GeneID# 1481778; UniProtKB/Swiss-Prot protein ID# Q7Y5R1) and mutants or derivatives thereof, which may be a catalytic domain selected from the group consisting of 5'→ Single-stranded RNA having sequence complementarity to the double-stranded template DNA in the 3' direction can be synthesized, and more particularly, it may be a catalytic domain of T7 RNA polymerase.

T7 RNA polymerase relates to the bacteriophage T7 DdRp. Preferably, the T7 RNA polymerase has the amino acid sequence of SEQ ID NO: 1 (NCBI genomic sequence ID# NC_001604; GeneID# 1261050; UniProtKB/Swiss-Prot ID# P00573) and is an 883 amino acid protein with a molecular weight of 98.8 kDa .

T7 RNA polymerase, in particular, in vitro, the enzyme is very processable and elongates 240-250 nucleotides/s at 37°C in the 5'→3' direction. Moreover, when expressed in eukaryotic cells, T7 RNA polymerase is mostly maintained in the cytoplasm and thus avoids the vigorous movement of large DNA molecules (i.e., transgenes) from the cytoplasm to the eukaryotic nucleus and export of RNA molecules from the nucleus to the cytoplasm. Optimize transgene expression levels.

The catalytic domain of DdRp can be one of the wild-type T7 RNA polymerase as well as the mutant of T7 RNA polymerase, which is single-stranded with sequence complementarity to the double-stranded template DNA in the 5'→3' direction even at reduced throughput. RNA can be synthesized. For example, the mutant may be selected from the group consisting of R551S, F644A, Q649S, G645A, R627S, I810S and D812E.

In the chimeric enzyme of the present invention, the catalytic domain of DdRp is located C-terminal to the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase and the catalytic domain of N7-guanine methyltransferase.

Indeed, when the catalytic domain of DdRp is the catalytic domain of a bacteriophage DdRp, in particular that of a bacteriophage DdRp selected from the group consisting of T7, T3 and SP6-RNA polymerase, said catalytic domain preferably preserves its native carboxyl terminus . In particular, the C-terminus of said catalytic domain of DdRp, in particular of said catalytic domain of bacteriophage DdRp, corresponds to the C-terminus of said chimeric enzyme. In particular, when the chimeric enzyme is a whole bacteriophage DdRp, in particular a bacteriophage DdRp selected from the group consisting of T7, T3 and SP6-RNA polymerases, said polymerase preferably preserves its native carboxyl-terminus. In particular, the C-terminus of said catalytic domain of DdRp, in particular of said catalytic domain of bacteriophage DdRp, corresponds to the C-terminus of said chimeric enzyme. In particular, said catalytic domain of DdRp, in particular said catalytic domain of bacteriophage DdRp selected from the group consisting of T7, T3 and SP6-RNA polymerase, is included in all or part of bacteriophage DdRp, wherein the C-terminus of said bacteriophage DdRp is said Corresponds to the C-terminus of the chimeric enzyme.

In the chimeric enzyme of the present invention, said catalytic domain of DdRp, in particular said catalytic domain of bacteriophage DdRp, in particular said catalytic domain of bacteriophage DdRp selected from the group consisting of T7, T3 and SP6-RNA polymerase, is said catalytic domain of RNA triphosphatase , located C-terminal to the catalytic domain of guanylyltransferase and the catalytic domain of N7-guanine methyltransferase.

The catalytic domain of DdRp is the catalytic domain of bacterial DdRp.

Preferably, the bacterial DdRp has a suitable structural complexity.

For example, the bacterial DdRp may be E. coli DdRp (NCBI genomic sequence of K-12 substrate DH10B ID# NC_010473), which has four different subunits (α subunit: rpoA GeneID# 6060938, UniProtKB). /Swiss-Prot ID# B1X6E7;β subunit: rpoB GeneID#6058462, UniProtKB/Swiss-Prot ID#B1XBY9;β' subunit: rpoC GeneID#6058956, UniProtKB/Swiss-Prot ID#B1XBZ0;σ subunit: rpoE GeneID# 6060683, UniProtKB/Swiss-Prot ID# B1XBQ0), assembled in a complex of five ααββ′σ subunits. The genomic elements involved in the regulation of enzymatic activity, including the E. coli RNA polymerase promoter, rho-dependent and -independent terminators and transcription stop sequences, have been well characterized.

The catalytic domain of DdRp is the catalytic domain of DdRp of eukaryotic cell phases, such as mitochondria, chloroplasts and proplasts. Indeed, these polymerases may also have a relatively simple structure.

The catalytic domain of DdRp may be the catalytic domain of mammalian mouse mitochondrial RNA polymerase, which is a single unit 120 kDa protein (GeneID# 216151, UniProtKB/Swiss-Prot ID# Q8BKF1) and shares homology with bacteriophage RNA polymerase. do. Several transcription factors are required for transcription initiation, elongation or termination: TFB1M (mitochondrial transcription factor Bl; mouse GeneID#224481, UniProtKB/Swiss-Prot ID# Q8JZM0) or TFB2M (mitochondrial transcription factor B2; mouse GeneID#) for transcriptional termination. 15278, UniProtKB/Swiss-Prot ID# Q3TL26), TFAM (mitochondrial transcription factor A; mouse GeneID# 21780, UniProtKB/Swiss-Prot ID# P40630), and mTERF (mitochondrial transcription terminator; mouse GeneID# 545725, UniProtKB/Swiss) -Prot ID# Q8CHZ9). The genomic elements involved in the regulation of the enzymatic activity of mitochondrial RNA polymerase, including transcription termination signals as well as two promoters in the light and heavy chains of the mitochondrial genome, have been well characterized.

RNA triphosphatase (RTPase) is an enzyme that removes the gamma phosphate residue at the 5' triphosphate terminus of the developing pre-mRNA to diphosphate.

RNA guanylyltransferase (GTase) refers to an enzyme that transfers GMP from GTP to the diphosphate-generating RNA terminus.

N7-guanine methyltransferase (N7-MTase) relates to an enzyme that adds a methyl residue on nitrogen 7 of guanine to the GpppN cap.

The catalytic domains of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase may be of the same or different capping enzymes. If the catalytic domains are from the same enzyme, then the catalytic domains of DdRp are from different enzymes.

Preferably, said catalytic domains of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase originate from one or several cytoplasmic enzymes, which advantageously have a relatively simple structure and well-characterized enzymatic activity. Thus, in particular, the catalytic domain of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase may be the catalytic domain of one or several viral capping enzymes, or capping enzymes of cytoplasmic episomes.

Said catalytic domains of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase may comprise one or several viral capping enzymes, in particular wild-type BTV capping enzyme, wild-type BMV capping enzyme, wild-type African swine fever virus capping enzyme, wild-type sub- Canthamoeba polyphaga is of an enzyme selected from the group consisting of a mimivirus capping enzyme and a mutant or derivative thereof, each of which is made into a diphosphate by removing the gamma phosphate residue at the 5' triphosphate terminus of the resulting pre-mRNA, or GMP can be transferred from GTP to the diphosphate generating RNA terminus, or a methyl residue on nitrogen 7 of guanine can be added to the GpppN cap, more particularly that of wild-type African swine fever virus capping enzyme.

The BTV capping enzyme relates to the single-unit VP4 capping enzyme of BTV (BTV), which is a 74 kDa protein (644 amino acids; for sequence, e.g., NCBI BTV serotype 10 genomic sequence ID# Y00421; GeneID# 2943157;UniProtKB/Swiss-Prot ID# P07132, D0UZ45, Q5J7C0, Q65751, Q8BA65, P33428, P33429, P33427, C3TUP7, Q8BAD5, C5IWW0, B4E551, Q3KVQ2, Q3KVP9) Q3 VP8, Q3K Q3KVQ3 Q3K Q3KVQ. This capping enzyme can homodimerize via a leucine zipper located adjacent to its carboxyl terminus. VP4 catalyzes all three enzymatic steps required for mRNA m7GpppN capping synthesis: RTPase, GTase and N7-MTase.

BMV enzyme relates to ORF1, BMV (BMV) mRNA capping enzyme, which is a single-unit 155-kDa protein (1365-amino acid; NCBI BMV isolate BaMV-0 genomic sequence ID# NC 001642; GeneID# 1497253; UniProtKB/ Swiss-Prot ID# Q65005). ORF1 protein has all the enzymatic activities required to produce m7GpppN mRNA capping, ie RTPase, GTase and N7-MTase. Furthermore, ORF1 has RNA-dependent RNA-polymerase activity, which is not governed by the chimeric enzyme activity of the present invention and can be disrupted by deletion of the Asp1229 Asp1230 residue of the mRNA capping enzyme. As used herein, the term "African swine fever virus capping enzyme" relates to NP868R capping enzyme (G4R), (ASFV), which is a single-unit 100 kDa protein (868 amino acids; NCBI ASFV genome sequence strain BA71V ID). # NC_001659; GeneID# 1488865; UniProtKB/Swiss-Prot ID# P32094).

The Acanthamoeba polyphaga mimivirus capping enzyme is directed to another single unit 136.5 kDa protein, R382 (APMV) (1170 amino acids; NCBI APMV genomic sequence ID# NC_006450; GeneID#3162607; UniProtKB/Swiss-Prot ID# Q5UQX1 ).

The catalytic domains of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase are of one or several capping enzymes of the cytoplasmic episome, such as pGKL2. In particular, the catalytic domains of RNA triphosphatase, guanylyltransferase and N7-guanine methyltransferase are included in all or part of the capping enzyme of the yeast linear extrachromosomal episomal pGKL2.

Cytoplasmic linear episomes are double-stranded DNA structures, which replicate stably in the cytoplasm of various yeast strains. One prototype of a yeast linear extrachromosomal episome, pgKL2 (13,457 bp; also termed pGK12), is Kluyveromyci lactis CB 2359 and Saccharomyci cerevisiae F102- were completely sequenced from various yeast strains, including 2. The capping enzyme encoded by the ORF3 gene of Kluyveromyces lactis pGKL2 (NCBI Kluyveromyces CB 2359 pGKL2 genomic sequence ID# NC_010187; UniProtKB/Swiss-Prot ID# P05469) is a 594 amino acid protein (70.6 kDa protein) )am.

In one embodiment of the chimeric enzyme of the invention, at least two, in particular at least three and more particularly the entire catalytic domain may be assembled, fused or linked directly or indirectly by a linking peptide.

In particular, at least two, in particular at least three and more particularly the entire catalytic domain selected from the group consisting of the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase, the catalytic domain of N7-guanine methyltransferase and DdRp are linked directly or by a linking peptide.

Linking peptides have the advantage of minimizing steric hindrance and providing sufficient space for the components of the fusion protein to create a fusion protein that maintains their native conformation.

Preferably, at least said catalytic domain of DdRp comprises said catalytic domain of RNA triphosphatase; the catalytic domain of guanylyltransferase; and the catalytic domain of N7-guanine methyltransferase is bound by a linking peptide to at least one catalytic domain selected from the group consisting of.

In particular, the linking peptide may be located N-terminal to said catalytic domain of DdRp, in particular said catalytic domain of bacteriophage DdRp selected from the group consisting of T7, T3 and SP6-RNA polymerase, said catalytic domain of RNA triphosphatase , C-terminal to the catalytic domain of guanylyltransferase and the catalytic domain of N7-guanine methyltransferase.

In particular, the catalytic domain of DdRp, in particular the N-terminus of the catalytic domain of a bacteriophage DdRp selected from the group consisting of T7, T3 and SP6-RNA polymerase, comprises the catalytic domain of RNA triphosphatase, said catalytic domain of guanylyltransferase connected to the C-terminus of one catalytic domain selected from the group consisting of a catalytic domain and said catalytic domain of N7-guanine methyltransferase by a covalent bond, in particular a linking peptide.

The linking peptide is a peptide of the formula (GlymSerp)n, wherein m is an integer from 0 to 12, in particular an integer from 1 to 8 and more particularly an integer from 3 to 6 and more particularly 4, and p is an integer from 0 to 6 , in particular an integer from 0 to 5, more particularly an integer from 0 to 3 and especially 1, n is an integer from 0 to 30, especially an integer from 0 to 12, more particularly an integer from 0 to 8 and even more particularly an integer from 1 to 6),

SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22 and a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 22.

A flexible linker peptide of formula (GlymSerp)n has the advantage that the glycine residue provides peptide flexibility and serine provides some solubility. Additionally, the absence of sensitive sites for kyrotrypsin I, factor Xa, papain, plasmin, thrombin and trypsin in the formula (GlymSerp)n linker sequence is thought to increase the overall stability of the resulting fusion protein.

Variable length formula (GlymSerp)n linkers are commonly used to construct single-stranded Fv fragment (sFv) antibodies. In addition, formula (GlymSerp)n linkers have been used to generate a variety of fusion proteins, which often retain the biological activity of each of their components.

Other types of peptide linkers are also chimeric enzymes according to the present invention, for example GGGGIAPSMVGGGGS (SEQ ID NO: 6), SPngASNSGSAPDTSSAPGSQ (SEQ ID NO: 7), EGKSSGSGSiKSTE (SEQ ID NO: 8), EGKSSGSGSiKEF (SEQ ID NO: 9), GGGSGGGSGGGTGGGSGGG (SEQ ID NO: 6) 10)], GSTSGSGKSSEGKG (SEQ ID NO: 11), YPRSIYIRRRHPSPSLTT (SEQ ID NO: 12), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 13), GSTSGSGKPGSGEGS (SEQ ID NO: 14), SSADDAKKDAAKKDDAKKKDDAKKDA (SEQ ID NO: 16), DDDLDAAKKDAAKKGS SEQ ID NO: LSDADKKKDAAKKGS SEQ ID NO: LSADDKKKDAAKKGS SEQ ID NO: l5), GSADDAKK 17), AEAAAKEAAAKEAAAKA (SEQ ID NO: 18), GSHSGSGKP (SEQ ID NO: 19), GSTSGSGKPGSGEGSTGAGGAGSTSGSGKPSGEG (SEQ ID NO: 20), LSLEVAEEIARLEAEV (SEQ ID NO: 21), and GTPTPTPTPTGEF (SEQ ID NO: 22).

Other types of covalent bonds include, but are not limited to, disulfide bonds, transglutamination and protein trans-binding by chemical and/or physical agents, such as tris(bipyridine)ruthenium(II)-dichloride and UV light irradiation.

Said catalytic domains of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase and DdRp can also be assembled by specific protein elements, for example leucine zippers, for example chimeric according to the invention In enzymes, the biotinylation domain can be assembled into one of the catalytic domains (eg, Avi-tag II or PFBtag) and the biotin binding domain into one of the other catalytic domains (eg, strep-tagII or nono-tag).

In the chimeric enzyme according to the invention, at least two of said catalytic domains may be assembled by non-covalent bonds, in particular by leucine zippers.

Preferably, at least said catalytic domain of DdRp is by a non-covalent bond, in particular a leucine zipper, the group consisting of said catalytic domain of RNA triphosphatase, said catalytic domain of guanylyltransferase and said catalytic domain of N7-guanine methyltransferase. It may be assembled into at least one of the catalytic domains selected from

Leucine zippers, a dimeric helical-coil protein structure composed of two amphoteric α-helices interacting with each other, are commonly used to homo- or hetero-dimerize proteins. Each helix consists of a repeat of 7 amino acids, wherein the first amino acid (residue a) is hydrophobic, the fourth amino acid (residue d) is usually leucine, and the other residues are polar. The leucine zippers VELCRO ACID-p1 and Base-p1, which form parallel heterodimeric double-stranded helical coil structures, have a high tendency to form parallel protein hetero-dimers. They have been used to heterodimerize several soluble proteins] as well as member proteins.

Other types of oligomerized peptide domains also produce chimeric enzymes according to the invention and form at least two of said domains of the chimeric enzymes according to the invention, in particular antiparallel heterodimeric structures, leucine zippers, for example ACID- It is thought to assemble al/Base-al, ACID-Kg/Base-Eg, NZ/CZ, ACID-pLL/Base-pLL and EE1234L and RR1234L leucine zippers. The disulfide-linked version of the leucine zipper can also be used to generate interchain disulfide bridges between cysteine residues under reducing conditions as well as in the disulfide helical coil-linked heterodimeric chimeric enzymes according to the present invention.

Thus, at least two of the catalytic domains of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase and DdRp are leucine zippers, in particular leucine zippers that form antiparallel heterodimeric structures, for example ACID- al/Base-al, ACID-Kg/Base-Eg, NZ/CZ and ACID-pLL/Base-pLL leucine zippers, disulfide helical coil-bonded zippers, and disulfide bridges between cysteine residues.

The chimeric enzyme of the present invention is, in particular, a wild-type T7 RNA polymerase capable of synthesizing single-stranded RNA having sequence complementarity to double-stranded template DNA in the 5'→3' direction via a linker and more particularly a linker of formula (Gly3Ser)4 or a wild-type mRNA capping enzyme of African swine fever virus or a mutant thereof, capable of adding an m7GpppN cap at the 5' end of the RNA molecule, fused to the amino terminus of a mutant or derivative thereof, in particular to the wild type of T7 RNA polymerase; or derivatives, particularly wild-type African swine fever virus capping enzymes.

The chimeric enzyme of the present invention is a wild-type T3 RNA polymerase capable of synthesizing single-stranded RNA having sequence complementarity to double-stranded template DNA in the 5'→3' direction, particularly through a linker and more particularly through a linker of formula (Gly3Ser)4 or a wild-type mRNA capping enzyme of African swine fever virus or a mutant thereof, capable of adding an m7GpppN cap at the 5' end of the RNA molecule, fused to the amino terminus of a mutant or derivative thereof, in particular to the wild type of T3 RNA polymerase, or derivatives, particularly wild-type African swine fever virus capping enzymes.

The chimeric enzyme of the present invention is, in particular, a wild-type SP6 RNA polymerase capable of synthesizing single-stranded RNA having sequence complementarity to double-stranded template DNA in the 5'→3' direction via a linker and more particularly a linker of formula (Gly3Ser)4 or a wild-type mRNA capping enzyme of African swine fever virus or a mutant thereof, capable of adding an m7GpppN cap at the 5' end of the RNA molecule, fused to the amino terminus of a mutant or derivative thereof, in particular to the wild type of SP6 RNA polymerase, or derivatives, particularly wild-type African swine fever virus capping enzymes.

The chimeric enzyme of the present invention is a wild-type mRNA capping enzyme of African swine fever virus or a mutant or derivative thereof, in particular wild-type African swine fever, capable of adding an m7GpppN cap at the 5' end of the RNA molecule, assembled by a leucine zipper. Virus capping enzyme and amino terminus of wild-type T7 RNA polymerase or a mutant or derivative thereof, in particular T7 RNA polymerase, capable of synthesizing single-stranded RNA with sequence complementarity to double-stranded template DNA in the 5'→3' direction. Includes wild type.

The chimeric enzyme in the present invention comprises at least one catalytic domain of the chimeric enzyme of the present invention, in particular the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase, the catalytic domain of N7-guanine methyltransferase and the catalytic domain of DdRp. It further comprises a domain enhancing the activity of at least one catalytic domain selected from the group consisting of.

For example, said domain, which enhances the activity of at least one catalytic domain of the chimeric enzyme of the present invention, has no intrinsic enzymatic activity but significantly inhibits the RNA N7-guanine methyltransferase activity of the D1R subunit of the vaccinia mRNA capping enzyme. It may be a 31-kDa subunit encoded by the vaccinia virus D12L gene, which enhances (genomic sequence ID#NC_006998.1; GeneID#3707515; UniProtKB/Swiss-Prot ID#YP_232999.1).

The chimeric enzymes of the present invention are, in particular, RNA-triphosphatase, RNA guanylyltransferase and RNA-guanine methyltransferase, assembled in whole or in part by a linker and more particularly by a linker of formula (Gly3Ser)4 and/or a leucine zipper. at least one catalytic domain of a vaccinia mRNA capping enzyme, having a enzyme activity, in particular a 95 kDa subunit encoded by the vaccinia virus D1R gene (Genomic sequence ID# NC 006998.1; GeneID# 3707562; UniProtKB/Swiss-Prot ID # YP 232988.1); - comprises at least one catalytic domain of DdRp, in particular a polymerase selected from the group consisting of T7, T3 and SP6-RNA polymerase and more particularly of T7 RNA polymerase and a 31-kDa encoded by the vaccinia virus D12L gene .

The invention also relates to an isolated nucleic acid molecule or group of isolated nucleic acid molecules, said nucleic acid molecule(s) encoding a chimeric enzyme according to the invention.

This group of isolated nucleic acid molecules encoding a chimeric enzyme of the invention comprises or consists of all nucleic acid molecules necessary and sufficient to obtain, by their expression, a chimeric enzyme according to the invention.

Said group of isolated nucleic acid molecules encoding a chimeric enzyme of the invention comprises at least one catalytic domain of RNA triphosphatase, at least one catalytic domain of guanylyltransferase and at least one catalytic domain of N7-guanine methyltransferase. It comprises or consists of a nucleic acid molecule encoding a nucleic acid molecule and a nucleic acid molecule encoding at least one catalytic domain of DdRp.

Said group of isolated nucleic acid molecules encoding a chimeric enzyme of the invention comprises a nucleic acid molecule encoding at least one catalytic domain of RNA triphosphatase, a nucleic acid molecule encoding at least one catalytic domain of guanylyltransferase, N7- comprises or consists of a nucleic acid molecule encoding at least one catalytic domain of guanine methyltransferase and a nucleic acid molecule encoding at least one catalytic domain of DdRp.

The nucleic acid molecule of the present invention may be functionally linked to at least one, preferably the entire promoter(s) selected from the group consisting of a promoter for eukaryotic DdRp, preferably RNA polymerase II, and a promoter for said catalytic domain of DdRp. can

The linkage of the nucleic acid to the promoter for eukaryotic DdRp, preferably RNA polymerase II, is markedly, when the chimeric enzyme of the present invention is expressed in a eukaryotic host cell, the expression of the chimeric enzyme is eukaryotic RNA polymerase, preferably It has the advantage of being induced by RNA polymerase II. These chimeric enzymes can also initiate transcription of a transgene. When a tissue-specific RNA polymerase II promoter is used, the chimeric enzyme of the present invention can be selectively expressed in a target tissue/cell.

The promoter may be a constitutive promoter or an inducible promoter known to those of ordinary skill in the art. The promoter may be developmentally regulated and may be inducible or tissue specific.

The present invention also relates to a vector comprising a nucleic acid molecule of the present invention. The vector may be suitable for semi-stable or stable expression.

The invention also relates to a group of vectors comprising said group of isolated nucleic acid molecules of the invention.

In particular, the vector of the present invention is a cloning or expression vector.

The vector may be a viral vector such as a bacteriophage or a non-viral vector such as a plasmid vector.

Said vector of the invention is, in particular, a bicistronic vector comprising a nucleic acid molecule of the invention and said catalytic domain of DdRp and/or a promoter for at least one DNA sequence of interest, said DNA sequence is operably linked to the promoter for the catalytic domain of DdRp.

The vector of the present invention may also comprise a promoter for the catalytic domain of DdRp.

The present invention also relates to an isolated nucleic acid molecule of the invention or a group of isolated nucleic acid molecules of the invention or a vector of the invention or a host cell comprising the group of vectors of the invention.

The host cells of the present invention may be useful for large-scale protein production.

Preferably, the catalytic domain of the DNA-polymerase RNA polymerase chimeric enzyme of the invention is of an enzyme different from that of the host cell to prevent competition between endogenous gene transcription and transgene transcription.

The present invention also relates to genetically engineered non-human eukaryotes expressing the chimeric enzymes of the present invention.

The non-human eukaryote may be any non-human animal and plant.

The present invention also relates to a chimeric enzyme according to the invention or an isolated nucleic acid molecule according to the invention or a group of isolated nucleic acid molecules according to the invention, in particular in vitro or It relates to use ex vivo.

The invention also relates to a chimeric enzyme according to the invention, in particular for the production of a protein, in particular an antibody, of a therapeutic purpose, such as a protein, in a eukaryotic system, for example in vitro synthesized protein assay or in cultured cells, to the in vitro or ex vivo use of an isolated nucleic acid molecule according to the present invention or a group of isolated nucleic acid molecules according to the present invention.

The present invention also relates to a particularly in vitro or ex vivo method for producing in a host cell an RNA molecule having a 5' terminal m7 GpppN cap encoded by a DNA sequence, said method comprising in a host cell a nucleic acid molecule according to the invention or the present invention expressing a group of isolated nucleic acid molecules according to the invention, wherein said DNA sequence is operably linked to a promoter for said catalytic domain of DdRp, in particular said promoter is effective in said host cell, in vitro or It relates to an ex vivo method.

Preferably, said catalytic domain of the DNA-dependent RNA-polymerase of the chimeric enzyme of the invention is of an enzyme different from that of the host cell to prevent competition between transcription of the endogenous gene and transcription of said DNA sequence.

Ⅲ. Modification of self-transcribed RNA/DNA systems

The DdRp RNA expression cassette, auto_DdRp expression cassette, DNAi expression cassette or auto_(DdRp+DNAi) expression cassette constituting the self-transcribed RNA/DNA system of the present invention may include at least one nucleotide sequence modification (SM).

The self-transcribed RNA/DNA system of the present invention is composed of RNA and DNA. The DdRp RNA expression cassette is a DdRp mRNA comprising a DdRp open reading frame (ORF), 5'UTR and 3'UTR, and may include a 5'cap structure, chemical modification (CM) and sequence modification (SM), If these symptoms were lacking in SM, they were called capped CM_DdRp mRNA, and if they were all included, it was called capped CM/SM_DdRp mRNA.

The "DdRp RNA expression cassette comprising capped CM/SM_DdRp mRNA" of the present invention is an expression cassette in which CM/SM_DdRp mRNA is prepared by including chemically modified nucleotides in SM_DdRp mRNA and a 5'cap structure is added. It is measured at a specific time point. The amount of protein or peptide produced from the capped CM/SM_mRNA was increased compared to SM_DdRp mRNA lacking CM (“capped SM_DdRp mRNA”). wherein the DdRp mRNA comprises at least one modification in the modification consisting of CM and SM that increases DdRp expression. The DdRp expression was further increased by administration of CM/SM_DdRp mRNA containing CM and SM. An increase in protein production according to at least one of CM/SM in capped CM/SM_DdRp mRNA is referred to herein as “CM/SM_DdRp mRNA-associated protein expression”.

The present invention provides a DdRp RNA expression cassette comprising elements managing the stability, replication, transcription and translation of the DdRp mRNA. If the DdRp RNA expression cassette method of the present invention is compared with the conventional plasmid DNA method having a nuclear-dependent promoter, the increase in protein production of the DdRp RNA expression cassette method is referred to herein as "DdRp RNA expression cassette-associated increase". mentioned.

The "DdRp RNA expression cassette-associated protein expression" of the present invention refers to a situation in which the protein produced from capped CM/SM_DdRp mRNA measured for a specific period is increased compared to the total amount of protein produced from reference DdRp mRNA lacking CM/SM. , or a situation in which the protein level produced from the capped CM/SM_DdRp mRNA measured at a specific time point is increased compared to the protein level produced from a reference DdRp mRNA lacking CM/SM.

In the present invention, the DNA of interest (DNAi) encodes an mRNA of interest (mRNAi) including at least one open reading frame (ORF), 5'UTR and 3'UTR encoding at least one peptide or protein, and a nucleotide sequence It may include a modification (SM), which was referred to as SM_DNAi. SM_DNAi of the DNAi expression cassette may encode SM_mRNAi.

In addition to this, the DdRp DNA of the auto_DdRp DNA expression cassette may be included, and a nucleotide sequence modification (SM) may be included, and was referred to as auto_DdRp DNA.

In the present invention, the amount of protein or peptide produced from SM_DNAi measured at a specific time point in the SM_DNAi coating mRNA with a nucleotide sequence modification (SM) also increases compared to DNA lacking the nucleotide sequence SM.

In the concept of the RNA/DNA system of the present invention, when a DNAi expression cassette comprising SM_DNAi along with the DdRp RNA expression cassette of the present invention is injected into cells/tissues, the expression of mRNAi encoded by the SM_DNAi is increased. The SM_DNAi includes at least one SM. The expression of the encoded protein or peptide is increased by administration of the mRNAi encoded by SM_DNAi. The increase in protein production following SM of DNA is referred to herein as "SM_DNA-associated protein expression".

According to the present invention, a SM_DNAi molecule is provided, characterized by increased expression of the encoded protein compared to the respective DNAi lacking SM (“reference DNAi”). To evaluate the in vivo protein production by SM_DNAi, the expression of the encoded protein is determined according to SM_DNAi into the target cell/tissue and compared with the protein expression of a reference DNAi.

According to the present invention, mRNAi DNA molecules can be SM to enhance the expression of the encoded protein (“SM_DNAi-associated increase”). Therein, SM is increased compared to a reference DNAi as defined herein without being limited to any particular structure as much as expression of the encoded protein. Several DNA variants are known in the art and can be applied to a given DNAi in the present invention.

The conventional method for expressing mRNA in the nucleus using a plasmid having a nuclear-dependent promoter depends on nuclear membrane penetration, which has an efficiency of only 1% compared to cell membrane penetration, and thus has very low RNA expression inhibition efficiency. In addition, the mRNA expressed in the nucleus can be synthesized into mRNA that can be involved in RNA expression only when it is released into the cytoplasm, but the exportin-5 transporter involved in the release into the cytoplasm is saturated by the high concentration of mRNA. The problem entails the problem of interfering with the cytoplasmic release of mRNA that is involved in other intracellular functions.

The self-transcription RNA/DNA system, which is a composition for continuous mRNAi expression of the present invention, is a DdRp RNA expression cassette containing the capped CM/SM_DdRp mRNA in a nuclear-independent manner by increasing the expression of T7 RNA polymerase selected from DdRp in the cytoplasm. It may be to continuously express the mRNAi expression located downstream of the T7 promoter of SM_DNAi in the mDdRp RNA expression cassette for a long period of time. Specifically, the sustained expression of the mRNAi may be a long-term continuous production of mRNAi in the cytoplasm from the composition, but is not limited thereto.

1. Modification of the 5' end (5' cap) of the modified RNA:

The DdRp RNA expression cassette, CM_mRNA, SM_mRNA and CM/SM_mRNA molecules of the present invention can be modified by the addition of a so-called "5' cap (CAP)" structure.

A 5' cap is usually a modifying entity that "caps" the 5' end of an entity, usually a mature mRNA. The 5' cap can be formed in general by modifications, in particular derivatives of guanine nucleotides. Preferably, the 5' cap is connected to the 5' end via a 5'5' triphosphate bond. The 5' cap may be methylated, for example, m7GpppN, where N is the 5' cap, usually the terminal 5' nucleotide of the nucleic acid carrying the 5' end of the RNA. m7GpppN is a naturally occurring 5' cap (CAP) structure in mRNA transcribed by polymerase II, and therefore is not considered as a SM included in CM/SM_mRNA according to the present invention. This means that the CM/SM_mRNA according to the present invention may comprise m7GpppN as the 5' cap (CAP), but additionally the SM_mRNAi comprises at least one additional SM as defined herein.

Examples of additional 5' cap structures include glyceryl, inverted deoxy abasic residues (components), 4',5' methylene nucleotides, 1-(beta-D-erythrofuranosyl)nucleotides, 4 '-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotide, alpha-nucleotide, chemically modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3' ,4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3'3' inverted nucleotide component, 3'3' inverted free base component, 3'2'-reversed nucleotide component, 3'2'-reversed base free component, 1,4-butanediol phosphate, 3' phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'phosphate, 3' phosphorothioate , phosphorodithioate, or a bridging or non-bridging methylphosphonate component. These chemically modified 5' cap (CAP) structures are considered to be at least one SM included in the SM_mRNAi according to the present invention.

Particularly preferred chemically modified 5' cap (CAP) structures are CAP1 (methylation of the ribose of the nucleotide adjacent to m7G), CAP2 (methylation of the ribose of the second nucleotide downstream of the m7G), CAP3 (the ribose of the third nucleotide downstream of m7G). methylation of), CAP4 (methylation of ribose downstream of the fourth nucleotide of m7G), ARCA (anti-reverse cap (CAP) analog, chemically modified ARCA (e.g. phosphothioate modified ARCA), inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

2. Chemical transformation (CM)

The term “RNA chemical modification” may refer to chemical modifications including sugar modifications or base modifications as well as backbone modifications.

The chemically modified RNA molecules of the present invention may contain nucleotide analogues/modifications, such as backbone modifications, sugar modifications or base modifications. A backbone modification in the context of the present invention is a modification in which the phosphate of the backbone of a nucleotide contained in a nucleic acid molecule as defined herein is chemically modified. A sugar modification in the context of the present invention is a modification of a sugar of a nucleotide of a nucleic acid molecule as defined herein. Moreover, a base modification in the context of the present invention is a modification of the nucleotide base component of a nucleic acid molecule of a nucleic acid molecule. Nucleotide analogues or variants are preferably selected from nucleotide analogues applicable to transcription and/or translation.

1) Sugar Modifications:

Chemically modified nucleosides and nucleotides that may be incorporated into the modified RNA of the present invention may be chemically modified in the sugar component. For example, the 2' hydroxyl group (OH) may be SM or substituted with a different number of "oxy" or "deoxy" substituents. Examples of "oxy"-2'hydroxyl group chemical modifications include, but are not limited to, alkoxy or aryloxy (-OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or Party); polyethylene glycol (PEG), -0(CH2CH2o)nCH2CH2OR; "locked" nucleic acids (LNAs) in which the 2' hydroxyl is linked to the 4' carbon of the same ribose sugar by, for example, a methylene bridge; An amino group (-O-amino, wherein the amino group, for example, NRR can be alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino ) or aminoalkoxy.

"Deoxy" variants include hydrogen, amino (eg, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or may be attached to the sugar via a linker, wherein the linker comprises an amino group comprising an atom, one or more of C, N and O.

A sugar group may also contain one or more carbons having the opposite stereochemical configuration to the corresponding carbon in ribose. Thus, SM_mRNA may comprise, for example, nucleotides containing arabinose as a sugar.

2) Backbone Modifications:

The phosphate backbone can also be modified with modified nucleosides and nucleotides that can be incorporated into modified RNA, as described herein. The phosphate group of the backbone can be modified by substituting one or more oxygen atoms with different substituents. In addition, modified nucleosides and nucleotides may include total substitution of a non-chemically modified phosphate moiety with a modified phosphate as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates. and phosphotriesters. Phosphorothioates have all unlinked oxygens substituted with sulfur. Phosphate linkers can also be SMed by substitution of nitrogen (bridged phosphoroamidate), sulfur (bridged phosphorothioate) and carbon (bridged methylene-phosphonate) in the linked oxygen.

3) base modifications:

As described herein, chemically modified nucleosides and nucleotides that can be incorporated into a modified RNA can be further SMed at a nucleobase moiety. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. For example, nucleosides and nucleotides as described herein can be chemically SM in the major groove face. The chemical modification may include an amino group, a thiol group, an alkyl group, or a halo group.

In the present invention, the nucleotide analogs/variants are selected from base variants, which are preferably 2-amino-6-chloropurineriboside-5'triphosphate, 2-aminopurine-riboside-5'triphosphate; 2-Aminoadenosine-5'triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'triphosphate, 2-thiouridine-5'triphosphate, 2' -Fluorothymidine-5'triphosphate, 2'-O-methyl inosine-5'triphosphate 4-thiouridine-5'triphosphate, 5-aminoallylcytidine-5'triphosphate, 5-aminoallyl Uridine-5'triphosphate, 5-bromocytidine-5'triphosphate, 5-bromouridine-5'triphosphate, 5-bromo-2'-deoxycytidine-5'triphosphate, 5-Bromo-2'-deoxyuridine-5'triphosphate, 5-iodocytidine-5'triphosphate, 5-iodo-2'-deoxycytidine-5'triphosphate, 5- iodouridine-5'triphosphate, 5-iodo-2'-deoxyuridine-5'triphosphate, 5-methylcytidine-5'triphosphate, 5-methyluridine-5'triphosphate, 5-propynyl-2'-deoxycytidine-5' triphosphate, 5-propynyl-2'-deoxyuridine-5' triphosphate, 6-azacytidine-5' triphosphate, 6-aza Uridine-5'triphosphate, 6-chloropurineriboside-5'triphosphate, 7-deazadenosine-5'triphosphate, 7-deazaguanosine-5'triphosphate, 8-azaadenosine-5' Triphosphate, 8-azidoadenosine-5'triphosphate, benzimidazole-riboside-5'triphosphate, N1-methyladenosine-5'triphosphate, N1-methylguanosine-5'triphosphate, N6-methyl adenosine-5'triphosphate, O6-methylguanosine-5'triphosphate, pseudouridine-5'triphosphate, or puromycin-5'triphosphate, xanthosine-5'triphosphate. of the base-CM consisting of 5-methylcytidine-5'triphosphate, 7-deazaguanosine-5'triphosphate, 5-bromocytidine-5'triphosphate, and pseudouridine-5'triphosphate Nucleotides for base SM selected from the group are particularly preferred.

Modified nucleosides include pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2- Thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudo Uridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl -uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2 -Thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

Modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethyl Tidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine Dean, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebrarin, 5-aza-zevurarin, 5-methyl-zeburarine, 5-aza-2-thio-zeburarine, 2-thio-zeburarine, 2-methoxy-cytidine, 2-methoxy-5-methyl-between tidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

The modified nucleosides are 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-de Aza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine , N6-Isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

Modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio -guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine , 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

The nucleotide may be SM and may comprise replacing the hydrogen at C-5 of uracil with a methyl group or a halo group.

Modified nucleosides are 5'0-(l-thiophosphate)-adenosine, 5'0-(1-thiophosphate)-cytidine, 5'0-(1-thiophosphate)-guanosine, 5'0 -(1-thiophosphate)-uridine or 5'0-(1-thiophosphate)-pseudouridine.

SM_mRNAi is 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl- Pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5- Methyl-uridine, pyrrolo-cytidine, inosine, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1 -Methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, a nucleoside chemical modification selected from the group consisting of:

4) Lipid modification:

The modified RNAs of the invention may contain lipid modifications. Such lipid-modified RNAs generally include RNAs as defined herein. Lipid-modified RNA molecules of the invention generally further comprise at least one linker covalently linked to the RNA molecule, and at least one lipid covalently linked to each linker. Instead, the lipid-modifying RNA molecule comprises at least one RNA molecule as defined herein and at least one (bifunctional) lipid covalently linked (without a linker) to the RNA molecule. According to a third alternative, the lipid-chemically modifying RNA molecule is an RNA molecule as defined herein, at least one linker covalently linked to the RNA molecule, and at least one lipid covalently linked to each linker, and also an RNA molecule and at least one (bifunctional) lipid covalently linked (without a linker). It is particularly preferred that the lipid modification is at the terminal end of the linear RNA sequence.

3. Modification of the base sequence (SM)

1) Variation of open reading frame: Variation of G/C content:

SM_mRNAi of the present invention encodes at least one peptide or protein, wherein the G/C content of the coding region is modified and is particularly increased when compared to the G/C content of its specific wild-type coding region, i.e., the unmodified coding region. . Preferably, the amino acid sequence of the coding region is not modified as compared to the encoded amino acid sequence of the particular wild-type coding region.

The modification of the G/C-content of the SM_mRNAi coding region in the present invention is based on the fact that the sequence of any mRNA region to be translated is important for efficient translation of the mRNA. Thus, the composition and sequence of the various nucleotides is important. In particular, an mRNA sequence having an increased content of G (guanosine)/C (cytosine) is more stable than an mRNA sequence having an increased content of A (adenosine)/U (uracil). According to the present invention, the codon of the coding region that is continuously being translated into the amino acid sequence is different from the codon of its wild-type coding region in that the amount of G/C nucleotides in the codon is increased. In light of the fact that several codons encode one and the same amino acid (the so-called degeneration of the genetic code), the codon most favorable for stability can be determined (the so-called alternative codon usage).

Depending on the amino acid to be encoded by the coding region of SM_mRNAi in the present invention, various possibilities exist for modification of the RNA sequence, eg, the coding region, when compared to its wild-type coding region. For amino acids encoded by codons containing only G or C nucleotides, no codon modifications are necessary. Therefore, since neither A nor U is present, the codons for Pro (CCC or CCG), Arg (CGC or CGG), ala (GCC or GCG) and Gly (GGC or GGG) do not require modification.

In contrast, codons containing A and/or U nucleotides may be modified by substitution of other codons encoding the same amino acid but not containing A and/or U, examples of which are:

The codon for Pro may be modified from CCU or CCA to CCC or CCG;

The codon for Arg may be modified from CGU or CGA or AGA or AGG to CGC or CGG;

The codon for ala can be modified from GCU or GCA to GCC or GCG;

The codon for Gly can be modified from GGU or GGA to GGC or GGG.

In other cases, although A or U nucleotides cannot be removed from the codon, the A and U content can be reduced by using codons containing lower amounts of A and/or U nucleotides. Examples of these are:

The codon for Phe can be modified from UUU to UUC;

The codon for Leu may be modified from UUA, UUG, CUU or CUA to CUC or CUG;

The codon for Ser can be modified from UCU or UCA or AGU to UCC, UCG or AGC;

The codon for Tyr can be modified from UAU to UAC;

The codon for Cys can be modified from UGU to UGC;

The codon for His can be modified from CAU to CAC;

The codon for Gln can be modified from CAA to CAG;

The codon for Ile can be modified from AUU or AUA to AUC;

The codon for Thr can be modified from ACU or ACA to ACC or ACG;

The codon for Asn can be modified from AAU to AAC;

The codon for Lys can be modified from AAA to AAG;

The codon for Val can be modified from GUU or GUA to GUC or GUG;

The codon for Asp can be modified from GAU to GAC;

The codon for Glu can be modified from GAA to GAG;

The stop codon UAA may be modified to UAG or UGA.

On the other hand, in the case of codons for Met(AUG) and Trp(UGG), there is no possibility of sequence modification.

In order to increase the G/C content of the coding region of SM_mRNAi in the present invention compared to the G/C content of a specific wild-type coding region (ie the original sequence), the substitutions listed above can be applied individually or in any possible combination. can be applied. Thus, for example, all codons for Thr occurring in the wild-type sequence can be modified to ACC (or ACG).

Preferably, in the present invention, the G/C content of the coding region of SM_mRNAi is increased by 7% or more, more preferably 15% or more, particularly preferably 20% or more, compared to the G/C content of the wild-type coding region. 5%, 10%, 20%, 30%, 40%, 50% of codons substitutable in the coding region encoding one or more peptides or proteins, including pathogenic antibodies or fragments, variants or derivatives thereof, 60% or more, more preferably 70% or more, even more preferably 80% or more, and most preferably 90% or more, 95% or more or even 100% are substituted, whereby the G/C content of said coding region is substituted. this will increase

Particularly preferably, in the present invention, the G/C content of the coding region of SM_mRNAi may increase by a maximum value (ie, 100% of substitutable codons) compared to the G/C content of the wild-type coding region.

2) Codon optimization:

A further preferred modification of the coding region encoding at least one peptide or protein of SM_mRNAi in the present invention is based on the finding that translation efficiency is also determined by different frequencies of appearance of tRNA in cells. Thus, if so-called "rare codons" are present to an increased degree in the coding region of a wild-type RNA sequence, they are translated to a significantly lower degree than if codons encoding a relatively "frequent" tRNA are present.

The coding region of the SM_mRNAi is preferably modified compared to the corresponding wild-type coding region so that the codons of the wild-type sequence encoding the tRNA, which are relatively rare in at least one cell, encode the tRNA that is relatively frequent in the cell and the relative is substituted with a codon carrying the same amino acid as the rare tRNA. By this modification, the coding region of the SM_mRNAi of the present invention is modified, and as a result, a codon for using a frequently occurring tRNA is inserted. In other words, according to the present invention, by this modification all codons of the wild-type coding region encoding a tRNA that are relatively rare in the cell can in each case be replaced with a codon encoding a tRNA that is relatively frequent in the cell, and , which in each case may carry amino acids as relatively rare tRNAs.

It is known to the person skilled in the art which tRNAs occur relatively frequently in cells and, in contrast, which tRNAs occur relatively rarely. Particular preference is given to the Gly codon using the most frequently occurring tRNA for a particular amino acid, for example the most frequently occurring tRNA in (human) cells.

It is particularly preferred to associate a maximally increased sequence G/C content in the coding region of the SM_mRNAi of the present invention with “frequent” codons in which the amino acid sequence of the peptide or protein encoded by the coding region of the RNA sequence is not modified. . Particularly efficiently translated and stabilized (chemically modified) RNA sequences are provided.

3) Variants of UTR:

Preferably, the SM_mRNAi of the present invention has at least one chemically modified 5' and/or 3' UTR sequence. Modifications in these 5' and/or 3' UTRs may have the effect of increasing the half-life of the RNA in the cell matrix or may increase the translation efficiency and thus enhance the expression of the encoded protein or peptide. These UTR sequences may have 100% sequence homology to naturally occurring sequences occurring in viruses, bacteria and eukaryotes, but may also be partially or fully synthesized.

The SM_mRNAi according to the present invention is preferably a 5' and/or 3' UTR element, in particular a 5'UTR element comprising or consisting of a nucleic acid derived from the 5'UTR of the TOP gene or from a fragment, homologue or variant thereof, or a stable mRNA 5' and/or 3'UTR elements, which may be derived from a gene providing a histone-stem-loop structure, preferably a histone-stem-loop in its 3' untranslated region; 5' cap (CAP) structure; poly-A tail; or at least one structural element from the group comprising a poly(C) sequence.

SM_mRNAi according to the present invention comprises at least one 5' or 3'UTR element. A UTR element comprises or consists of a nucleic acid sequence derived from any naturally occurring 5' or 3'UTR, or derived from a fragment, homologue or variant of a 5' or 3'UTR of a gene. Preferably, the 5' or 3'UTR element used according to the invention is heterologous to the coding region of the SM_mRNAi according to the invention. Although 5' or 3'UTR elements derived from naturally occurring genes are preferred, synthetically engineered UTR elements may also be used in the present invention.

The sequence of SM_mRNAi according to the present invention comprises or consists of at least one 5' untranslated region element (5 'UTR element).

The nucleic acid sequence of the 5'UTR element derived from the 5'UTR of the TOP gene is 1, 2, 3, 4, 5, 6, upstream of the start codon (eg, A(U/T)G) of the gene or mRNA. It terminates at its 3' end with a nucleotide located at position 7, 8, 9 or 10. Thus, the 5'UTR element does not include any portion of the protein coding region. Thus, preferably, only the protein coding portion of the SM_mRNAi according to the present invention is provided by the coding region.

The nucleic acid sequence derived from the 5'UTR of the TOP gene is derived from a eukaryotic TOP gene, preferably a plant or animal TOP gene, more preferably a chordate TOP gene, even more preferably a vertebrate TOP gene, most preferably a It is preferably derived from a mammalian TOP gene, such as a human TOP gene.

SM_mRNAi according to the present invention is derived from the 3'UTR of a chordid gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene, or a chordate gene, preferably a vertebrate gene, More preferably it further comprises at least one 3'UTR element comprising or consisting of a nucleic acid sequence derived from a variant of the 3'UTR of a mammalian gene, most preferably a human gene.

The term '3'UTR element' refers to a nucleic acid sequence comprising or consisting of a nucleic acid sequence derived from a 3'UTR or derived from a variant of the 3'UTR. In the sense of the present invention, a 3'UTR element may represent the 3'UTR of an mRNA. Thus, in the sense of the present invention, preferably, the 3'UTR element may be the 3'UTR of an mRNA, preferably of an artificial mRNA, which may also be an electron template for the 3'UTR of an mRNA. Thus, the 3'UTR element is preferably a nucleic acid sequence corresponding to the 3'UTR of an mRNA, preferably an artificial mRNA, such as a 3'UTR of an mRNA obtained by transcription of a genetically engineered vector expression cassette. Preferably, the 3'UTR element realizes the function of the 3'UTR or encodes a sequence that realizes the function of the 3'UTR.

A novel mRNA comprises a 3'UTR element that can be derived from a gene associated with an mRNA with improved half-life (providing a stable mRNA), for example a 3'UTR element as defined and described below.

The 3'UTR element is a gene selected from the group consisting of an albumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, such as a collagen alpha 1(I) gene. from the 3'UTR of or from the group consisting of albumin gene, α-globin gene, β-globin gene, tyrosine hydroxylase gene, lipoxygenase gene, and collagen alpha gene, such as collagen alpha 1 (I) gene. It comprises or consists of a nucleic acid sequence derived from a variant of the 3'UTR of the gene of choice. The 3'UTR element may be an albumin gene, preferably a vertebrate albumin gene, more preferably a mammalian albumin gene,

Most preferably it comprises or consists of a nucleic acid sequence derived from the 3'UTR of the human albumin gene.

Human Albumin 3'UTR: CATCACATTT AAAAGCATCT CAGCCTACCA TGAGAATAAG AGAAAGAAAA TGAAGATCAA AAGCTTATTC ATCTGTTTTT CTTTTTCGTT GGTGTAAAGC CAACACCCTG TCTAAAAAAC ATAAATTTCT TTAATCATTT TGCCTCTTTT CTCTGTGCTT SEQ ID NO: 6)

It is particularly preferred that the SM_mRNAi according to the present invention comprises a 3'UTR element comprising a corresponding RNA derived from another nucleic acid or a fragment, homologue or variant thereof.

Most preferably, the 3'UTR element comprises a nucleic acid sequence derived from a fragment of the human albumin gene according to SEQ ID NO:6:

Albumin7 3'UTR: CATCACATTT AAAAGCATCT CAGCCTACC ATGAGAATAA GAGAAAGAAA ATGAAGATCA ATAGCTTATT CATCTCTTTT TCTTTTTCGT TGGTGTAAAG CCAACACCCT GTCTAAAAAA CATAAATTTC GGTTTAATCATT TTGCCTATCTTT GAACCT (SEQ ID NO: 7) TCTCTGTGCT TCAAT.

XRMGL preferably, the 3'UTR element of the inventive mRNA comprises or consists of the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO:6.

The 3'UTR element is an α-globin gene, preferably a vertebrate α- or β-globin gene, more preferably a mammalian α- or β-globin gene, most preferably a human α according to SEQ ID NOs: 6-7 - or comprises or consists of a nucleic acid sequence derived from the 3'UTR of the β-globin gene:

3'UTR of Homo sapiens hemoglobin, alpha 1 (HBA1): GCTGGAGCCT CGGTGGCCAT GCTTCTTGCC CCTTGGGCCT CCCCCCAGCC CCTCCTCCCC TTCCTGCACC CGTACCCCCG TGGTCTTTGA ATAAAGTCTG AGTGGGCGGC (SEQ ID NO: 8)

3'UTR of Homo sapiens hemoglobin, alpha 2 (HBA2): GCTGGAGCCT CGGTAGCCGT TCCTCCTGCC CGCTGGGCCT CCCAACGGGC CCTCCTCCCC TCCTTGCACC GGCCCTTCCT GGTCTTTGAA TAAAGTCTGA GTGGGCAG (SEQ ID NO: 9)

3'UTR of Homo sapiens hemoglobin, beta (HBB): GCTCGCTTTC TTGCTGTCCA ATTTCTATTA AAGGTTCCTT TGTTCCCTAA GTCCAACTAC TAAACTGGGG GATATTATGA AGGGCCTTGA GCATCTGGAT TCTGCCTAAT AAAAAACATT TATTTTCATT GC (SEQ ID NO:10)

For example, the 3'UTR element may preferably comprise or consist of a central, α-complex-binding portion of the 3'UTR of an α-globin gene, such as a human α-globin gene according to SEQ ID NO:7:

central, the α-complex-binding portion of the 3′UTR of the α-globin gene (also termed herein as “muag”); GCCCGATGGG CCTCCCAACG GGCCCTCCTC CCCTCCTTGC ACCG (SEQ ID NO: 11).

It is particularly preferred that the 3'UTR element of SM_mRNAi according to the present invention comprises or consists of a corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 11 or a homologue, fragment or variant thereof.

Preferably, the at least one 5'UTR element and the at least one 3'UTR element play a synergistic role in increasing the production of the SM_mRNAi derived protein according to the invention as described above. More preferably, the at least one 5'UTR element and/or the at least one 3'UTR element plays a synergistic role with the injection of SM_mRNAi to increase protein production.

SM_mRNAi according to the present invention may further comprise - by adding at least one modification - an internal ribosome entry site (IRES) sequence or an IRES-motif, for example, if SM_mRNAi is two or more Encoding peptides or proteins can be separated into several open reading frames. The IRES-sequence can be particularly helpful if the mRNA is a bi- or multicistronic RNA.

As used herein, the term “RNA” is not limited and refers to any ribonucleic acid. Thus, the term RNA applies equally to ribonucleic acids that function as coding RNAs, for example as viral RNA, (replicon) or mRNA, or to artificial DdRp RNA expression cassettes of any type.

According to the present invention, mRNAi comprises at least one modification that increases the expression of the peptide or protein encoded by the coding region of the RNA. The mRNAi according to the invention comprises at least one type of modification as defined herein. Instead, or in combination with other types of modifications, certain modifications as defined herein, such as 3'UTR, histone stem-loop or poly C sequence, may also be present in one or more copies in the SM_mRNAi according to the invention. can This is especially true for bi- or multicistronic RNAs. SM_mRNAi according to the present invention may comprise a combination of several distinct modifications, eg, any, eg 5'UTR, poly C sequence, poly A sequence, 3'UTR or histone stem-loop and GC- Modifications that combine GC-enrichment, wherein each distinct modification may exist in the form of a single copy per RNA or multiple copies per RNA.

Moreover, for SM_mRNAi comprising at least one open reading frame to be administered, the 3' region of the coding region may be modified, for example, by insertion or addition of multiple repeat stretches of adenine or cytosine residues. The 3' end of the RNA molecule is chemically modified by the addition of a serii of adenine nucleotides ("poly(A) tail").

The length of the poly(A) tail has a major influence on the translation efficiency of each constituting the expression cassette. To be efficiently translated, the poly(A) tail of an exogenously delivered mRNA must consist of at least 20 A residues. Furthermore, mRNA expression has been described to correlate positively with the length of the poly(A) tail (Tavernier et al., J Control Release. 2011 Mar 30;150(3):238-47). Shows improved mRNA stability and translation efficiency compared to shorter poly(A) tails measured with 120 nucleotides (Holtkamp et al., Blood. 2006 Dec 15;108(13):4009-17) . SM_mRNAi of the present invention comprise (optionally in combination with other modifications) a poly(A) tail, wherein the poly(A) tail has at least 30, preferably greater than 50, more preferably 100 poly(A) tails. more than, even more preferably more than 200 adenosine nucleotides. Most preferably, the RNA comprises a poly(A) tail consisting of 64 adenosine nucleotides.

If the poly(A) tail is longer than 30 adenosines, preferably longer than 50 adenosines, more preferably longer than 100 adenosines, and even more preferably longer than 200 adenosines, the poly(A) tail is only It is considered as at least one modification included in SM_mRNAi according to the present invention.

Modification of the poly(A) tail is not considered to be at least one modification of SM_mRNAi according to the present invention. This means that the SM_mRNAi according to the present invention may comprise a poly(A) tail as defined above, but may comprise at least one further modification of the present invention.

SM_mRNAi comprising at least one open reading frame comprises (optionally in combination with other modifications) a poly(C) sequence in region 3' of the coding region of the RNA. The poly(C) sequence generally comprises a plurality of cytosine nucleotides, generally between about 10 and about 200 cytidine nucleotides, preferably between about 10 and about 100 cytidine nucleotides, more preferably between about 10 and about 70 cytosine nucleotides. a stretch of tidine nucleotides or even more preferably from about 20 to about 50 or even from about 20 to about 30 cytidine nucleotides. The poly(C) sequence may preferably be located 3' of the coding region by the nucleic acid. The poly(C) sequence of the present invention is located 3' to the poly(A) sequence.

SM_mRNAi according to the present invention comprises or encodes a histone stem-loop at its 3' end as at least one modification.

A particularly preferred embodiment for a histone stem-loop sequence is the sequence according to CAAAGGCTCTTTTCAGAGCCACCA (SEQ ID NO: 12) or the corresponding RNA sequence according to CAAAGGCUCUUUUCAGAGCCACCA (SEQ ID NO: 13).

IV. mRNA of interest (mRNAi)

The mRNAi of the present invention may be any one selected from the group consisting of antigens, antibodies, therapeutic proteins, peptides and fusion proteins. The mRNAi comprises at least one open reading frame encoding a therapeutic protein or peptide. Antigens such as pathogenic antigens, tumor antigens, allergens or autoimmune antigens are encoded by at least one open reading frame. Therein, administration of mRNAi encoding an antigen is used in a genetic vaccination approach against a disease comprising the antigen. The antibody is encoded by at least one open reading frame of SM_mRNAi according to the invention.

The mRNAi according to the present invention does not include a reporter gene or a marker gene. Preferably, SM_mRNAi according to the present invention is, for example, luciferase; green fluorescent protein (GFP) and variants thereof (eg, eGFP, mRNAi or BFP); α-globin; hypoxanthine-guanine phosphoribosyltransferase (HGPRT); β-galactosidase; galactokinase; alkaline phosphatase; It does not encode secreted embryonic alkaline phosphatase (SEAP) or resistance genes (eg, neomycin, puromycin, hygromycin and zeocin). The mRNAi according to the invention does not encode luciferase. The mRNAi according to the present invention does not encode GFP or a variant thereof.

fusion protein

A fusion protein is a protein composed of at least two domains or more encoded by separate genes that are transcribed and translated as a single unit and joined to produce a single polypeptide. There are basically two types of fusion proteins. The first consists of two proteins or protein subunits that are fused end-to-end and are usually linked by a linker and the second have the amino acids of the two donors interspersed in the fusion protein.

The former is a heterologous protein polypeptide fused with a linker peptide; And it may be any one selected from the group comprising; and a special-purpose peptide and a polypeptide bound to a protein.

Fusion proteins can be classified according to the protein domain into which they are integrated. Often one fusion partner has a molecular recognition function while the other may have specific functions such as improved stability and half-life, cytotoxic effects or novel targeting and delivery pathways. Therapeutic fusion proteins can be immunotoxins, cytokine fusions, fragment crystallizable (Fc) fusions, albumin fusions and others not belonging to these classes. Although most ongoing clinical trials for recombinant proteins involve monoclonal antibodies, fusion proteins are expected to become important therapeutic molecules in the future.

Pathogenic antigens:

The mRNAi according to the present invention may encode a protein or peptide comprising a pathogenic antigen or a fragment, variant or derivative thereof. Such pathogenic antigens are derived from pathogenic organisms, in particular bacteria, viruses or protozoan (multicellular) pathogenic organisms, which elicit an immune response in a subject, in particular a mammalian subject, more particularly a human. More specifically, preferably the pathogenic antigen is a surface antigen, eg a protein (or a fragment of a protein, eg an external portion of a surface antigen) located on the surface of a virus or bacterial or protozoan organism.

Pathogenic antigens are preferably selected from antigens derived from pathogens associated with infectious diseases. Particularly preferred antigens are influenza virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), human papillomavirus (HPV), human immunodeficiency virus (HIV), Plasmodium, Staphylococcus aureus), Dengue virus, Chlamydia trachomatis, cytomegalovirus (CMV), hepatitis B virus, Mycobacterium tuberculosis, rabies virus and yellow fever virus. am.

The mRNAi according to the present invention encodes a rabies virus protein or peptide or antigenic fragment thereof. Preferably, the mRNAi according to the present invention is a rabies virus glycoprotein G (RAV-G), nucleoprotein N (RAV-N), phosphoprotein P (RAV-P), matrix protein M (RAV-M) or RNA polymer It encodes an antigenic protein or peptide selected from the group consisting of Rase L (RAV-L), or a fragment, variant or derivative thereof.

The mRNAi according to the present invention encodes a respiratory syncytial virus (RSV) protein or peptide or antigenic fragment thereof. Preferably, SM_mRNAi according to the present invention is a respiratory syncytial virus (RSV) fusion protein F, glycoprotein G, short hydrophobic protein SH, matrix protein M, nucleoprotein N, large polymerase L, M2-1 protein, M2 -2 encodes an antigenic protein or peptide selected from the group consisting of protein, phosphoprotein P, non-structural protein NS1 or non-structural protein NS2, or a fragment, variant or derivative thereof.

Tumor antigens:

The mRNAi according to the present invention may encode a tumor antigen, a peptide comprising a fragment, variant or derivative of the tumor antigen, or a protein or peptide comprising the protein, and is an antigen or a fragment, variant or derivative of the tumor antigen.

Oncogene (KRAS, p53, etc.) mutant cancer antigen, tumor antigen NY-iO-1, 5T4, MAGE-C1, MAGE-C2, Survivin, Muc-1, PSA, PSMA, PSCA, STEAP and PAP are particularly preferred. The administered mRNAi encodes a protein or peptide comprising a therapeutic protein or a fragment, variant or derivative thereof.

The therapeutic protein of the present invention is a peptide or protein that is beneficial for the treatment of any inherited or acquired disease or improves the condition of an individual. In particular, therapeutic proteins play a large role in the creation of therapeutics, and can modify and repair genetic errors, destroy cancer cells or pathogen-infected cells, treat immune system disorders, and treat metabolic or endocrine disorders, among other functions. For example, erythropoietin (EPO), a protein hormone, may be used to treat patients with red blood cell deficiency, a common cause of kidney complications. Adjuvant proteins, therapeutic antibodies, are also encompassed by therapeutic proteins and hormone replacement therapy, which can be used, for example, in the treatment of postmenopausal women. In a more recent approach, a patient's somatic cells are used to dedifferentiate into pluripotent stem cells to replace disputed stem cell therapies. Also, those proteins used in the retrodifferentiation of somatic cells or used in the differentiation of stem cells are defined herein as therapeutic proteins. In addition, the therapeutic protein may be used for other purposes such as, for example, wound healing, tissue regeneration, angiogenesis, and the like.

Thus, therapeutic proteins can be, for example, infectious diseases, neoplasms (eg cancer or tumor diseases), blood and blood-forming organ diseases, endocrine, nutritional and metabolic diseases, neurological diseases, circulatory system diseases, respiratory system diseases. It can be used for a variety of purposes, including the treatment of a variety of disorders (independently hereditary or acquired) such as diseases of the digestive system, diseases of the skin and subcutaneous tissue, diseases of the musculoskeletal system and connective tissue, and diseases of the genitourinary system.

Among them, a particularly preferred therapeutic protein that can be used in the treatment of metabolic or endocrine disorders is understood to be a therapeutic protein since it is intended to treat a subject by replacing its insufficient internal production of a functional protein with a sufficient amount. Accordingly, such therapeutic proteins are generally mammalian, in particular human proteins. Therapeutic proteins can be used for the treatment of blood disorders, circulatory disorders, respiratory disorders, cancer or tumor disorders, infectious disorders or immunodeficiency disorders:

Furthermore, adjuvants or immune stimulating proteins are also encompassed by the term therapeutic protein. An adjuvant or immune stimulating protein can be used to treat a specific disease or alleviate a condition of a subject by inducing, modifying, or improving an immune response in a subject. Adjuvant proteins are mammalian, in particular human adjuvant proteins, generally including any human protein or peptide capable of inducing an innate immune response (in mammals), for example, in response to the binding of an exogenous TLR ligand to a TLR. can be selected from is selected from the group consisting of proteins such as The mammalian, particularly human adjuvant protein may also be selected from the group consisting of a heat shock protein, or any species homologue of any of such human adjuvant proteins. Mammalian, particularly human adjuvant proteins may also include proteins that induce or enhance an innate immune response.

therapeutic protein

Therapeutic proteins or adjuvant proteins for the treatment of blood disorders, diseases of the circulatory system, diseases of the respiratory system, cancer or tumor diseases, infectious diseases or immunodeficiency are generally proteins of mammalian origin, preferably of human origin, depending on the animal to be treated. The human subject is preferably treated, for example, with a therapeutic protein of human origin.

Pathogenic adjuvant proteins generally include pathogenic adjuvant proteins capable of inducing an innate immune response (in mammals), more preferably pathogenic adjuvant proteins derived from bacteria, protozoa, viruses or fungi, etc.; For example, a bacterial (adjuvant) protein, a protozoan (adjuvant) protein (eg, a propylin-like protein of Toxoplasma gondii), a viral (adjuvant) protein, or a fungal (adjuvant) and pathogenic adjuvant proteins selected from proteins and the like.

Bacterial (adjuvant) proteins may also include bacterial flagellin. Protozoan (adjuvant) proteins are further examples of pathogenic adjuvant proteins. The protozoan (adjuvant) protein may be selected from any protozoan protein exhibiting adjuvant properties, and the viral (adjuvant) protein is another example of a pathogenic adjuvant protein. The viral (adjuvant) protein may be selected from any viral protein exhibiting adjuvant properties, and more preferably, but not limited to, a Respiratory Syncytial Virus fusion glycoprotein (F-protein). , MMT virus-derived envelope protein, mouse leukemia virus protein, wild-type measles virus hemagglutinin protein, and the like may be selected from the group consisting of. Fungal (adjuvant) proteins are further examples of pathogenic adjuvant proteins. In the present invention, the fungal (adjuvant) protein may be selected from any fungal protein exhibiting adjuvant properties, more preferably, fungal immunomodulatory protein (FIP; LZ-8), etc. may be selected from the group. Finally, the adjuvant protein may further be selected from the group consisting of keyhole-limpet hemocyanin (KLH), OspA, and the like.

Therapeutic proteins may be used in hormone replacement therapy, particularly in the treatment of postmenopausal women. These therapeutic proteins are preferably selected from oitrogens, progesterones or progitins, and often testosterone.

Moreover, therapeutic proteins can be used to reverse differentiate somatic cells into pluripotent or omnipotent stem cells. For this purpose, several factors, inter alia, Oct-3/4, the Sox gene family (Sox1, Sox2, Sox3 and Sox15), the Klf family (Klf1, Klf2, Klf4 and Klf5), the Myc family (c-myc, L-myc) and N-myc), Nanog and LIN28.

A therapeutic antibody is also defined herein as a therapeutic protein. These therapeutic antibodies are inter alia antibodies used for the treatment of cancer or oncological diseases, inter alia antibodies used for the treatment of blood disorders, inter alia antibodies used for immunomodulation, antibodies used for the treatment of diabetes, antibodies used for the treatment of Alzheimer's disease Antibodies, may include antibodies used to treat asthma,

The coding region of SM_mRNAi according to the present invention may occur as mono-, di-, or even multicistronic RNA, ie RNA carrying the coding sequence of one, two or more proteins or peptides. Coding sequences in such di-, or even multicistronic RNAs can induce cleavage of a polypeptide comprising, for example, at least one internal ribosome entry site (IRES) or several proteins or peptides as described herein. can be separated by a signal peptide.

V. Expression cassettes and transporters

The self-transcribed RNA/DNA system of the present invention includes the contents of the novel expression cassette expressed by DdRp, and in more detail, the developed linear DdRp RNA expression cassette, auto_DdRp expression cassette, DdRp expression system expressed by auto-loop , a DdRp promoter and a DNAi expression cassette encoding the mRNA of interest, an auto_(DdRp+DNAi) expression cassette, an antibiotic resistance gene, an origin of replication and a recombinant expression cassette comprising the cloning site sequence and transfection with the expression cassette It relates to transformed cells and the like.

Restriction endonuclease is present in almost all bacteria and is an enzyme that recognizes a specific nucleotide sequence present in DNA and cuts the recognized sequence site or a specific site near it. These restriction enzymes are originally possessed by bacteria as a defense mechanism to remove foreign DNA such as viruses that invade their cells, and their DNA is protected from restriction enzymes by methylating the base (adenine or cytosine) at the cleavage site. are doing By using the properties of these restriction enzymes, a desired gene can be inserted into the vector.

In particular, the expression cassette has a multicloning site (MCS), which artificially collects the recognition sites of various restriction enzymes and determines the restriction enzyme of the insert DNA for cloning. Plasmid vectors are commercially available by constructing MCS mainly from restriction enzymes commonly used. In MCS using such general-purpose restriction enzymes, there are genes in which it is difficult or impossible to clone all genes due to their high frequency.

The recombinant expression cassette of the present invention is a cassette capable of expressing a target protein or target RNA in a host cell, and refers to a gene construct including essential regulatory elements operably linked to express a gene insert. The expression cassette includes an expression control sequence and, if necessary, a signal sequence or leader sequence for membrane targeting or secretion, and can be prepared in various ways depending on the purpose. The expression control sequence refers to a DNA sequence that controls the expression of an operably linked polynucleotide sequence in a specific host cell. Such regulatory sequences include promoters to effect transcription, optional operator sequences to control transcription, 5'Cap structures, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation. The expression cassette may also include a selection marker for selecting a host cell comprising the cassette and, in the case of a replicable expression cassette, an origin of replication.

The recombinant expression cassette of the present invention may include a nucleotide sequence encoding various known proteins, and the nucleotide sequence encoding the target protein is operably linked with a promoter. To be operably linked means that one nucleic acid fragment is associated with another nucleic acid fragment so that its function or expression is affected by the other nucleic acid fragment.

The expression cassette according to the present invention can be prepared by inserting the MCS polynucleotide of the present invention as a basic backbone to an existing cassette used for protein expression so as to be operably linked by a promoter to which a DdRp selected for each structural gene binds. there is. Preferably, the expression cassette of the present invention may include an origin of replication, a promoter, a fluorescent protein gene, MCS, and a selection marker, and may further include a polyA signal sequence for protein expression.

In the present invention, the selectable marker (selective marker) may be an antibiotic resistance gene, and many genes that can be such an antibiotic resistance gene are currently known (eg, kanamycin, neomycin, ampicillin, tetracycline, etc.) . Accordingly, a person skilled in the art can select and use a desired gene from the known antibiotic resistance genes, and the specific kind of such gene does not constitute the essence of the present invention. In addition, since the introduction of the antibiotic resistance gene is for selecting transformants, any gene that can be used for selection may be introduced. Therefore, it is not limited to the antibiotic resistance gene. Preferably, the antibiotic resistance gene of the present invention may be a kanamycin or neomycin resistance gene.

The expression cassette of the present invention also includes an expression cassette in which the base sequence of the expression cassette of the present invention is slightly changed according to a method known to those skilled in the art, such as codon degeneracy, as long as the basic structure of the expression cassette of the present invention is the same or similar. In addition, it is apparent to those skilled in the art that the N-terminus and the C-terminus of the sequence listing form a circle.

On the other hand, the present invention provides a cell transformed with the recombinant cassette of the present invention.

The recombinant expression cassette of the present invention can be introduced into a host using methods known in the art. Examples include, but are not limited to, calcium chloride and heat shock methods, particle gun bombardment, silicon carbide whiskers, sonication, electroporaion, PEG- Medium fusion method, microinjection method, microinjection method, liposome-mediated method, vacuum infiltration method, etc. can be used.

The host cell may be cells of animals, plants, yeast and bacteria, preferably, in the case of other than bacteria, it can well accept a vector introduced from the outside, and the boundary between intracellular organelles such as the cytoplasm and the nucleus is clearly distinguished. Cells are preferred. More preferably, CHO-k1 (ATCC CCL-61, Cricetulus griseus, hamster, Chinie), HEK293 (ATCC CRL-1573, Homo sapiens, human), HeLa (ATCC CCL-2, Homo sapiens, human), SH-SY5Y (ATCC CRL-2266, Homo sapiens, human), Swiss 3T3 (ATCC CCL-92, Mus musculus, mouse), 3T3-L1 (ATCC CL-173, Mus musculus, mouse), NIH/3T3 (ATCC CRL-1658, Mus musculus, mouse), L-929 (ATCC CCL-1, Mus musculus, mouse), Rat2 (ATCC CRL-1764, Rattus norvegicus, rat), RBL-2H3 (ATCC CRL-2256, Rattus norvegicus, rat), MDCK (ATCC CCL-34, Canis familiaris). In addition, it may be various other stem cells, cells extracted from various tissues, and artificially made similar cell membrane structures.

Meanwhile, standard recombinant DNA and molecular cloning techniques used in the present invention are well known in the art and are described in the following literature (Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual) , 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989); by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience (1987)).

The present invention provides an improved expression cassette comprising the above-described nuclear-independent cytoplasmic gene expression control sequence by DdRp.

In the present invention, the expression cassette of the present invention is a plasmid vector such as E. coli-derived plasmid, Bacillus subtilis-derived plasmid, yeast-derived plasmid and Ti plasmid, cosmid vector, adenovirus (Lockett LJ, et al., Clin. Cancer Ri. 3:2075-2080 (1997)), adeno-associated virusi (AAV) (Lashford LS., et al., Gene Therapy Technologii, Applications and Regulations Ed. A. Meager (1999)), retro Viruses (Gunzburg WH, et al., Gene Therapy Technologii, Applications and Regulations Ed. A. Meager, (1999)), lentiviruses (Wang G. et al., J. Clin. Invit. 104(11):R55- 62 (1999)), herpes simplex virus (Chamber R., et al., Proc. Natl. Acad. Sci USA 92:1411-1415 (1995)), vaccinia virus (Puhlmann M. et al., Human Gene) Therapy 10:649-657 (1999)), reovirus, poxvirus (GCE, NJL, Krupa M, iteban M., The poxvirus vectors MVA and NYVAC as gene delivery systems for vaccination against infectious diseasi and cancer Curr Gene Ther 8 ( 2):97-120(2008)), Semliki Forester Virus, Polymer (Hwang et al., In vitro and In vivo Transfection Efficiency of Human Osteoprotegerin Gene using Non-Viral Polymer Carriers., Korean J. Bone Metab. 13 (2):11 9-128 (2006)), liposomes (Methods in Molecular Biology, Vol 199, S.C. Basu and M. Basu (Eds.), Human Pris 2002), can be applied to nanomaterials or niosomes.

1. Plasmids

Plasmids are circular DNA molecules that exist independently in bacterial cells. Plasmids almost always contain more than one gene, and these genes are often responsible for useful characteristics exhibited by the host bacteria. For example, the ability to survive at normal toxic concentrations of antibiotics is due to the presence of plasmids with antibiotic resistance genes in bacteria. In the laboratory, this antibiotic resistance is often used as a selectable marker.

All plasmids have at least one DNA sequence that can serve as an origin of replication (a site recognizable by the replication enzyme of the host cell, from which replication begins), and thus the main bacterial chromosome ( It can replicate in cells independently of the main bacterial chromosome). Some types of plasmids can replicate by inserting themselves into the bacterial chromosome. These integrating plasmids (called episomes) are able to maintain their conformation stably during numerous cell divisions. However, at some stage, it exists as an independent element by excision (the reverse process of the process of being integrated).

A selectable marker is a gene carried by a vector, and refers to a gene that enables recognition of the characteristics of a cell containing this vector or recombinant DNA molecule.

The size of the plasmid ranges from 1.0 kb for the smallest to more than 250 kb for the largest. Copy number refers to the number of plasmids normally found in one bacterial cell. The factors controlling the copy number are not well understood, but each plasmid has a characteristic number of 1 to 50 or more.

Although plasmids are widely distributed in bacteria, they are by no means so common in other organisms. The most studied eukaryotic plasmid exists in yeast as a 2 μm circle. The discovery of the 2 μm plasmid was very fortunate as it made possible the preparation of vectors for cloning the genes of industrial organisms of great importance as hosts. However, no plasmid has been found in other eukaryotes.

2. Adenovirus

Adenovirus has been widely used as a gene delivery vector because of its medium genome size, ease of manipulation, high titer, wide range of target cells, and excellent infectivity. Both ends of the genome contain 100-200 bp inverted terminal repeat (ITR), which is an essential cis element for DNA replication and packaging. The E1 regions of the genome (E1A and E1B) encode proteins that regulate transcription and transcription of host cell genes. The E2 region (E2A and E2B) encodes proteins involved in viral DNA replication. Among the currently developed adenovirus vectors, replication-deficient adenoviruses lacking the E1 region are widely used. On the other hand, the E3 region is removed in conventional adenoviral vectors to provide a site for insertion of a foreign gene (Thimmappaya, B. et al., Cell, 31:543-551 (1982); and Riordan, J. R. et al., Science, 245:1066-1073 (1989)). Therefore, the gene expression control sequence of the present invention can be inserted into the promoter sequence position of the E1A gene to regulate the expression of the E1A gene. In addition, the gene expression control sequence-expression gene cassette of the present invention is preferably inserted into the deleted E1 region (E1A region and/or E1B region, preferably E1B region) or E3 region, more preferably the deleted E1 region inserted into the area. In addition, the gene expression control sequence-expression gene cassette of the present invention can be inserted into the deleted E4 region. As used herein, the term "deletion" in relation to a viral genome sequence has a meaning including a partial deletion as well as a complete deletion of the corresponding sequence.

In addition, since adenovirus can pack up to about 105% of the wild-type genome, it can additionally package about 2 kb (Ghosh-Choudhury et al., EMBO J., 6:1733-1739 (1987)). Accordingly, the above-described foreign sequence inserted into the adenovirus may additionally bind to the genome of the adenovirus.

The foreign gene carried by the adenovirus is replicated in the same manner as the episome, and thus has very low genetic toxicity to the host cell. Therefore, it is judged that gene therapy using the adenovirus gene delivery system of the present invention is very safe.

3. Retrovirus

Retroviruses are widely used as gene delivery vectors because they insert their genes into the host genome, can transport a large amount of foreign genetic material, and have a wide spectrum of cells that can infect.

In order to construct a retroviral vector, the target nucleotide sequence to be transported is inserted into the retrovirus genome instead of the retrovirus sequence to produce a replication-incompetent virus. To produce virions, a packaging cell line containing the gag, pol and env genes but lacking the long terminal repeat (LTR) and Ψ sequences was constructed (Mann et al., Cell, 33:153-159 (1983)). When a recombinant plasmid containing the target nucleotide sequence to be transported, LTR and Ψ sequence is introduced into the cell line, the Ψ sequence enables production of an RNA transcript of the recombinant plasmid, which is packaged into a virus, and the virus is excreted into the medium (Nicolas and Rubinstein "Retroviral vectors," In: Vectors: A survey of molecular cloning vectors and their usi, Rodriguez and Denhardt (eds.), Stoneham: Butterworth, 494-513 (1988)). The medium containing the recombinant retrovirus is collected, concentrated and used as a gene delivery system.

Gene transfer using a second-generation retroviral vector has been published. Kasahara et al. Science, 266:1373-1376 (1994)) prepared a mutant of Moloney murine leukemia virus, in which an EPO (erythropoietin) sequence was inserted into the envelope region to produce a chimeric protein having novel binding properties. The gene delivery system of the present invention can also be prepared according to the construction strategy of the second-generation retroviral vector.

4. AAV Vectors

Adeno-associated virus (AAV) is suitable as the gene delivery system of the present invention because it can infect non-dividing cells and has the ability to infect various types of cells. Detailed descriptions of the preparation and use of AAV vectors are disclosed in detail in US Pat. Nos. 5,139,941 and 4,797,368. A study of AAV as a gene delivery system is described in LaFace et al, Viology, 162:483486 (1988), Zhou et al., Exp. Hematol. (NY), 21:928-933 (1993), Walsh et al, J. Clin. Invit., 94:1440-1448 (1994) and Flotte et al., Gene Therapy, 2:29-37 (1995). Recently, AAV vectors are being conducted clinically I as a therapeutic agent for cystic fibrosis.

Typically, the AAV virus is a plasmid (McLaughlin et al., J. Virol., 62:1963) containing the target gene sequence (relaxin gene and the target nucleotide sequence to be transported) flanked by two AAV terminal repeats. 1973 (1988); and Samulski et al., J. Virol., 63:3822-3828 (1989)) and an expression plasmid (McCarty et al., J. Virol. , 65:2936-2945 (1991)).

5. Other Virus Vectors

Other viral vectors can also be used in the gene delivery system of the present invention. Basinia virus (Puhlmann M. et al., Human Gene Therapy 10:649-657 (1999); Ridgeway, "Mammalian exprision vectors," In: Vectors: A survey of molecular cloning vectors and their usi. Rodriguez and Denhardt, eds Stoneham: Butterworth, 467-492 (1988); Baichwal and Sugden, "Vectors for gene transfer derived from animal DNA virusi: Transient and stable exprision of transferred geni," In: Kucherlapati R, ed. Gene transfer. New York: Plenum Pris, 117-148 (1986) and Coupar et al., Gene, 68:1-10 (1988)), lentivirus (Wang G. et al., J. Clin. Invit. 104(11):R55-62) (1999)) or herpes simplex virus (Chamber R., et al., Proc. Natl. Acad. Sci USA 92:1411-1415 (1995)), reovirus, poxvirus (GCE, NJL, Krupa M, iteban) M., The poxvirus vectors MVA and NYVAC as gene delivery systems for vaccination against infectious diseasi and cancer Curr Gene Ther 8(2):97-120(2008)) It can be used as a delivery system capable of transporting the sequence into a cell.

6. Polymer

Polymeric carriers widely used as non-viral gene carriers include gelatin, chitosan (Carreno GB, Duncan R. Evaluation of the biological propertii of soluble chitosan and chitosan microspheri. Int J Pharm 148:231-240(1997)) PLL ( poly-LLysine) (Maruyama A, Ishihara T, Kim JS, Kim SW, Akaike T. Nanoparticle DNA carrier with poly (L-lysine) grafted polysaccharide copolymer and poly (D,Llactide). Bioconjugate Chem 8:735-742(1997) )) and polyethyleneamine (PEI) (Abdallah B, Hassan A, Benoist C, Goula D, Behr JP, Demeneix BA. A powerful nonviral vector for in vivo gene transfer into the adult mammalian brain: Polyethyleneimine. Human Gene Ther 7:1947- 1954 (1996)) are being used. The advantages of polymer-based gene carriers are that the expression rate of immune response and acute toxicity is low, the preparation method is simple, and mass production is possible.

7. Liposomes and niosomes

Liposomes are automatically formed by phospholipids dispersed in an aqueous phase. Examples of successful intracellular delivery of foreign DNA molecules as liposomes are described by Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190 (1982) and Nicolau et al., Methods Enzymol., 149:157-176 (1987). Meanwhile, Lipofectamine (Gibco BRL) is the most widely used reagent for transfection of animal cells using liposomes. Liposomes containing the target nucleotide sequence to be transported interact with cells through mechanisms such as endocytosis, adsorption to the cell surface, or fusion with plasma cell membranes to deliver the target nucleotide sequence to be transported into the cell.

While liposomes are made using phospholipids, niosomes are used and polar and non-polar substances can be encapsulated with a double membrane transporter composed of a mixture of nonionic surfactant and cholesterol. , osmotically active, more stable than liposomes, which are carriers of lipid particles by phospholipids, etc., and also have the advantage that the surfactant used in the preparation does not require special conditions for storage and handling.

8. Nanomaterials

As used herein, the term nanomaterial refers to a biocompatible polymer material having a size of several to several hundreds of nanometers, for example, polyethylene glycol (polyetheleneglycol, PEG), polylactide (poly-lactide, PLA), polyglycol Lead (polyglycolide, PGA), polylactide-co-glycolide (PLGA), polycaprolactone (poly-ε-carprolactone, PCL), hyaluronic acid (HA), chitosan (chitosan) ), or serum albumin.

Meanwhile, the method of introducing the gene delivery system of the present invention into a cell as described above can be carried out through various methods known in the art.

In the present invention, when the gene delivery system is constructed based on a viral vector, it is carried out according to a viral infection method known in the art. Infection of host cells with viral vectors is described in the references cited above.

When the gene delivery system in the present invention is a naked recombinant DNA molecule or plasmid, the microtransformation method (Capecchi, M. R., Cell, 22:479 (1980); and Harland and Weintraub, J. Cell Biol. 101: 1094-1099 (1985)), calcium phosphate precipitation (Graham, F.L. et al., Virology, 52:456 (1973); and Chen and Okayama, Mol. Cell. Biol. 7:2745-2752 (1987)), Electroporation (Neumann, E. et al., EMBO J., 1:841 (1982); and Tur-Kaspa et al., Mol. Cell Biol., 6:716-718 (1986)), liposome-mediated Transfection (Wong, T.K. et al., Gene, 10:87 (1980); Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190 (1982); and Nicolau et al., Methods Enzymol., 149: 157-176 (1987)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190 (1985)), and gene bambadment (Yang et al., Proc. Natl. Acad. Sci) ., 87:9568-9572 (1990)), the gene can be transfected into cells.

Ⅵ. pharmaceutical composition

Use of a composition comprising a plurality of self-transcribed RNA/DNA systems of the present invention for the preparation of a pharmaceutical composition for increasing the expression of an encoded peptide or protein, in particular for example, preferably a gene for the treatment of a disease as defined herein A composition comprising a plurality of self-transcribed RNA/DNA systems of the invention, for example for therapeutic or genetic vaccination, is administered to a cell (eg, an expression host cell or somatic cell), tissue or organism, preferably It relates to the use for the preparation of a pharmaceutical composition to be applied or administered by jet injection as a pharmaceutical composition as described herein, either in naked form or in complex form or as a pharmaceutical composition as described herein.

A pharmaceutical composition comprising the self-transcribed RNA/DNA system of the present invention, or a composition comprising a plurality of the self-transcribed RNA/DNA system of the present invention, and optionally also a pharmaceutically acceptable carrier and/or vehicle .

As a first component, the pharmaceutical composition comprises at least one chemically modified nucleic acid as defined herein.

As a second component, the pharmaceutical composition may optionally comprise at least one additional pharmaceutically active component. A pharmaceutically active ingredient related thereto is a compound having a therapeutic effect of curing, alleviating or preventing certain indications or diseases as mentioned herein. Such compounds include, but are not implied to be limited thereto, preferably a peptide or protein as defined herein, preferably a nucleic acid as defined herein, a (therapeutically active) low molecular weight organic or inorganic compound (molecular weight less than 5000; preferably less than 1000), preferably sugars, antigens or antibodies as defined herein, therapeutic agents already known in the prior art, antigenic cells, antigenic cell fragments, cell fractions; cell wall components (eg polysaccharides), chemically modified, attenuated or inactivated (eg, chemically or by irradiation) pathogens (viruses, bacteria, etc.), preferably as defined herein adjuvants and the like.

In addition, the pharmaceutical composition may include a pharmaceutically acceptable carrier and/or vehicle. In the present invention, a pharmaceutically acceptable carrier generally comprises a liquid or non-liquid basis of the inventive pharmaceutical composition. The carrier is generally pyrogen-free water, isotonic saline or buffered (aqueous) solutions, for example, phosphate, citrate, etc. buffered solutions. Reference media may be, for example, a liquid present in an "in vivo" method, such as blood, lymph, cytosolic liquid, or other bodily fluid, or, for example, a conventional buffer or A liquid that can be used as a reference medium in "in vitro" methods, such as liquids. Such conventional buffers or liquids are known to the person skilled in the art. Ringer-Lactate solution is particularly preferred as a liquid basis.

However, one or more compatible solid or liquid fillers or diluents or encapsulating compounds may also be used as pharmaceutical compositions suitable for administration to the patient to be treated. As used herein, the term "compatible" means in such a way that the constituents of these pharmaceutical compositions do not interact under normal conditions of use to substantially reduce the pharmaceutical effectiveness of the pharmaceutical composition. It means that chemically modified RNA can be mixed.

Complexation:

In addition, the pharmaceutical composition may include a carrier for the self-transcribed RNA/DNA system of the present invention. Such carriers may be suitable for dissolution in physiologically acceptable liquids, pharmaceutically, for mediating transport and cellular uptake of the RNA/DNA systems of the invention. Accordingly, such a carrier may be a component suitable for storage and transport of the RNA/DNA system of the present invention according to the present invention. Such a component may be, for example, a cationic or polycationic carrier or compound which may serve as a transforming or complexing agent.

Particularly preferred transforming or complexing agents are cationic or polycationic compounds such as protamine, nucleoline, spermine or spermidine, or other cationic peptides or proteins such as poly-L-lysine ( PLL), poly-arginine, basic polypeptides, cell penetrating peptides (CPPs), such as HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, penetratin, VP22 derived or similar peptides; HSV VP22 (Herpi simplex), MAP, KalA or protein transforming domains (PTDs), PpT620, proline-rich peptide, arginine-rich peptide, lysine-rich peptide, MPG-peptide(s), Pep-1 , L-oligomer, calcitonin peptide(s), Antennapedia-derived peptide (especially from Drosophila antennapedia, pAntp, pIsl, FGF, lactoferrin, transportan, bufferin- 2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptide, SAP, or histone.

In addition, such a cationic or polycationic compound or carrier may be a cationic or polycationic peptide or protein, and may preferably contain or be further modified to include at least one -SH component. Preferably, the cationic or polycationic carrier is selected from cationic peptides having the general formula (III):

{(Arg)l(Lys)m(His)n(Orn)o(Xaa)x}; Formula (III)

where l+m+n+o+x = 3 - 100, and independently of each other l, m, n or o is the total amount of Arg (arginine), Lys (lysine), His (histidine) and Orn (ornithine) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 when representing at least 10% of the total amino acids of the oligopeptide , 19, 20, 21 - 30, 31 - 40, 41 - 50, 51 -60, 61 - 70, 71 - 80, 81 - 90 and 91 - 100, and Xaa is Arg(Arginine ), Lys (lysine), His (histidine) or Orn (ornithine); x is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, if the total amount of Xaa does not exceed 90% of the total amino acids of the oligopeptide 15, 16, 17, 18, 19, 20, 21 - 30, 31 - 40, 41 - 50, 51 - 60, 61 - 70, 71 - 80, 81 - 90. Any amino acid of Arg, Lys, His, Orn and Xaa may be located at any position in the peptide. Cationic peptides or proteins in the range of 7 to 30 amino acids are particularly preferred.

In addition, according to formula {(Arg)l(Lys)m(His)n(Orn)o(Xaa)x} (formula (III)) As defined, cationic or polycationic peptides or proteins are selected from, but not limited to, subformula (Ia):

{(Arg)l(Lys)m(His)n(Orn)o(Xaa')x (Cys)y} subformula (IIIa)

where (Arg)l(Lys)m(His)n(Orn)o and x are as defined herein, Xaa' is any amino acid selected from natural (= naturally occurring) or non-natural amino acids except Arg, Lys, His, Orn or Cys, if the total amount represents at least 10% of the total amino acids of the oligopeptide, y is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21- 30, 31 - 40 , 41 - 50, 51 - 60, 61 - 70, 71 - 80 and 81 - 90. In addition, the cationic or polycationic peptide may be selected from subformula (IIIb):

Cys1 {(Arg)l(Lys)m(His)n(Orn)o(Xaa)x} Cys2 Subformula (IIIb)

wherein the empirical formula {(Arg)l(Lys)m(His)n(Orn)o(Xaa)x} (formula (IV)) is as defined herein, and (semiempIREScal)) Forming the core of the amino acid sequence according to , Cys1 and Cys2 are proximal or terminal cysteines of (Arg)l(Lys)m(His)n(Orn)o(Xaa)x.

Further preferred cationic or polycationic compounds which can be used as transformation or complexing agents are cationic polysaccharides such as chitosan, polybrene, cationic polymers such as polyethyleneimine (PEI), Cationic lipids such as DOTMA: [1-(2,3-thioreyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC -Chol, BGTC, CTAP, DOPC, DODAP, DOPE: dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS: dioctadecylamidoglycylspermine, DIMRI: dimyristo-oxypropyl dimethyl Hydroxyethyl ammonium bromide, DOTAP: dioleyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride , CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy) )ethyl]trimethylammonium, CLIP9: rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine, or cationic or polycationic polymers such as , β-amino acid-polymers or chemically modified polyamino acids such as reversed polyamides, chemically modified polyethylenes such as PVP (poly(N-ethyl-4-vinylpyridinium bromide)), pDMAEMA (poly(dimethylamino Chemically modified acrylates such as ethylmethylacrylate)), chemically modified amidoamines such as pAMAM (poly(amidoamine)), diamine ends chemically modified 1,4 butanediol diacrylate-co- Chemically modified polybetaaminoesters (PBAE) such as 5-amino-1-pentanol polymer, dendrimers such as polypropylamine dendrimers or pAMAM-based dendrimers, PEI: poly(ethyleneimine), poly(propyleneimine), etc. Polyimine(s), polyallylamine, cyclodextrin-based polymers, dextran-based polymers, sugar backbone-based polymers such as chitosan, silane backbone-based polymers such as PMOXA-PDMS copolymers, one or more cationic blocks (eg, one selected from the above-mentioned cationic polymers) and block polymers composed of one or more hydrophilic or hydrophobic blocks (eg, polyethylene glycol) and the like.

It is particularly preferred that the RNA/DNA system of the present invention is at least partially complexed with a cationic or polycationic compound, preferably a cationic protein or peptide. In part, it means that only part of the SM_mRNAi molecule is complexed with a cationic or polycationic compound and the rest of the SM_mRNAi molecule is in an uncomplexed form (“free”). Preferably, the ratio of complexed SM_mRNAi to free SM_mRNAi ranges from about 5:1 (w/w) to about 1:10 (w/w), more preferably from about 4:1 (w/w) to from about 1:8 (w/w), even more preferably from about 3:1 (w/w) to about 1:5 (w/w) or 1:3 (w/w); , most preferably the ratio of complexed nucleic acid to free nucleic acid is selected from a ratio of about 1:1 (w/w).

The pharmaceutical composition may include an adjuvant. An adjuvant may be understood as any compound suitable for initiating or increasing the immune response of the innate immune system, ie, a non-specific immune response. In other words, upon administration, the inventive pharmaceutical composition preferably induces an innate immune response, optionally according to the adjuvant contained therein. Preferably, such adjuvants may be selected from adjuvants known to the person skilled in the art and may be suitable in the present case, ie supporting the induction of an innate immune response in a mammal, for example as defined above an adjuvant protein as defined below or an adjuvant as defined below.

Particularly preferred as adjuvants suitable for storage and delivery are cationic or polycationic compounds as defined above for the RNA/DNA systems of the invention as vehicles, transformation or complexing agents.

Additional additives that may be included in the inventive pharmaceutical composition include emulsifiers, such as, for example, twin wetting agents, such as, for example, sodium lauryl sulfate; coloring agent; flavoring agents, pharmaceutical carriers; tablet-forming agents; stabilizers; antioxidants; There are preservatives.

The pharmaceutical composition also has a binding affinity (as a ligand) to the Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or murine Any additional known immunostimulatory due to binding affinity (as ligand) to the murine toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13 It may further contain a compound.

The pharmaceutical composition can preferably be administered subcutaneously, intramuscularly, or intradermally. Sterile injectable forms of the pharmaceutical compositions may be aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.

A pharmaceutical composition generally comprises a "safe and effective amount" of the components of a pharmaceutical composition, in particular the RNA/DNA system of the present invention. As used herein, "safe and effective amount" means an amount of an RNA/DNA system of the invention as defined herein sufficient to significantly induce a positive modification of a disease or disorder as defined herein. do. At the same time, however, a “safe and effective amount” is small enough to avoid serious side effects and low enough to allow for a sensible relationship between advantage and risk. Determination of such limitations is generally within the scope of reasonable medical judgment.

Also of the self-transcribed RNA/DNA system of the invention, of a composition comprising a plurality of self-transcripting RNA/DNA systems of the invention as defined herein, of a pharmaceutical composition comprising the RNA/DNA system of the invention, or multiple applications and uses of kits comprising the same.

The present invention relates to the self-transcribed RNA/DNA system of the inventive invention as defined herein or the self-transcribed RNA/DNA system as defined herein, preferably as a medicament, in particular in gene therapy or genetic vaccination, for the treatment of a disease as defined herein. It relates to the primary medical use of the inventive composition comprising a plurality of self-transcribed RNA/DNA system molecules of the inventive invention as described above.

The present invention relates to the secondary medical use of the self-transcribed RNA/DNA system of the present invention, or the inventive composition for the treatment of a disease as defined herein comprising a plurality of molecules of the self-transcribed RNA/DNA system of the present invention and preferably the RNA/DNA system of the present invention, the inventive composition comprising a plurality of molecules of the self-transcribed RNA/DNA system of the present invention, a pharmaceutical composition comprising the same, or a kit comprising the same as defined herein It relates to the use for the manufacture of a medicament for the prevention, treatment and/or alleviation of a disease as described above. Preferably, the pharmaceutical composition is used or administered to a patient in need thereof for this purpose.

Preferably, the diseases of the present invention are infectious diseases, neoplasms (eg cancer or tumor diseases), blood and blood-forming organ diseases, endocrine, nutritional and metabolic diseases, diseases of the nervous system, diseases of the circulatory system, diseases of the respiratory system, diseases of the digestive system, diseases of the skin and subcutaneous tissue, diseases of the musculoskeletal system and connective tissue and diseases of the genitourinary system.

In the present invention, the term *?**?*carrier *?**?* is included without limitation as long as it is a substance that enhances the delivery of the RNA/DNA system into cells. Specifically, the carrier may be a liposome, a polymer-based nanoparticle, a dendrimer, a gold nanoparticle, and more specifically, a cationic liposome generally used for genetic material delivery. , The liposome is DC-Chol/DOPE liposome, DMRIE/DOPE liposome, EDMPC/Chol liposome, GL-67/DOPE/DMPE/PEG liposome, or DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine) liposome may be, but is not limited thereto.

In addition, the carrier is the self-transcribed RNA/DNA system of the present invention, and may be delivered into a cell to perform a DdRp expression loop to increase DdRp expression in the cytoplasm and thus RNA expression.

In the present invention, the carrier may be characterized in that it selectively accumulates in cancer tissue. The cancer is lung cancer, stomach cancer, colon cancer, breast cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, head and neck cancer, melanoma, uterine cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, perianal cancer, fallopian tube carcinoma, endometrial cancer, Cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine adenocarcinoma, prostate cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, lymphocytic lymphoma , kidney cancer, ureter cancer, renal pelvic cancer, blood cancer, brain cancer, central nervous system (CNS) tumor, spinal cord tumor, brainstem glioma or one or more cancers selected from the group consisting of pituitary adenoma, but may be limited thereto doesn't happen

Provided is a method for preparing the mRNAi transporter in order to achieve the target. capped CM_DdRp mRNA: auto_DdRp expression cassette; And the self-transcription RNA/DNA system including the DNAi expression cassette and the contents of the delivery system are the same as described above.

Specifically, the preparation method comprises the steps of preparing capped CM_DdRp mRNA, auto_DdRp expression cassette and DNAi expression cassette, respectively; and mixing the DOPC with the RNA / DNA system comprising the capped CM_DdRp mRNA, the auto_DdRp expression cassette and the DNAi expression cassette, further comprising the step of removing the free RNA / DNA system by filtration after the mixing step may include, but is not limited to.

In the mixing step, the self-transcription RNA/DNA system and DOPC may be mixed in a ratio of 1:5 to 1:10 (w/w), and more specifically, prepared by mixing in a ratio of 1:10. can, but is not limited thereto. In addition, the DNAi expression cassette: capped CM_DdRp mRNA; auto_DdRp expression cassette = may be mixed in a molar ratio of 0.4:1:1, but is not particularly limited thereto.

The present invention relates to a self-transcribed RNA/DNA system comprising a DdRp RNA expression cassette and a DNAi expression cassette or a composition comprising an RNA/DNA system comprising the auto_DdRp expression cassette in addition to the self-transcribed RNA/DNA system. It can be used for the preparation of a composition, in particular for the purpose as defined herein, preferably for gene therapy or genetic vaccination to treat diseases as defined herein.

Alternatively, the preparation method comprises the steps of preparing a capped CM_DdRp mRNA and auto_(DdRp+DNAi) expression cassette, respectively; and mixing the DOPC with the RNA/DNA system comprising the capped CM_DdRp mRNA and auto_(DdRp+DNAi) expression cassette, and further removing the free RNA/DNA system by filtration after the mixing step may include, but is not limited thereto.

The pharmaceutical composition may also be used for gene therapy or genetic vaccination, particularly preferably for the treatment of a disease or disorder as defined herein.

According to a further aspect, the present invention also provides kits, in particular kits of parts.

Such kits, in particular kits of parts, generally comprise either component alone or in combination with a pharmaceutical composition or vaccine comprising an additional component as defined herein, at least one RNA/DNA system as defined herein. The at least one RNA/DNA system as defined herein is optionally combined with a further component as defined herein, whereby the at least one self-transcribed RNA/DNA system according to the invention comprises one or more other components. provided separately from at least one other part of the kit (the first part of the kit).

The pharmaceutical composition may be present, for example, in one or different parts of the kit. By way of example, for example, at least one part of the kit may comprise at least one RNA/DNA system as defined herein, and at least one additional part of the kit, and at least one other component as defined herein. may, for example, other parts of at least one kit may comprise at least one pharmaceutical composition or parts thereof, for example, at least one part of the kit may comprise an RNA/DNA system of the invention, at least one additional kit part at least one other component as defined herein, at least one additional kit part at least one pharmaceutical composition component or the entire pharmaceutical composition, and at least one additional kit part for example , at least one pharmaceutical carrier or vehicle, and the like. In case the kit or kit of parts comprises a plurality of molecules of the self-transcribed RNA/DNA system of the present invention, one component of the kit may include only one, several or all SM_mRNAi molecules or the corresponding DNA contained in the kit. . All/each RNA/DNA systems of the invention may be included in different/separate components of the kit and each component forms part of the kit. In addition, one or more other (second, third, etc.) components (provided in one or more other kits of parts) may contain one or more SM_mRNAi, which may be identical or partially identical to or different from the first component, One or more RNA/DNA systems of the invention may be included in the first component as part of a kit. A kit or kit of parts may also include the administration and administration of the RNA/DNA system of the invention, the pharmaceutical composition, or any component thereof or, for example, the parts when the kit is prepared as a kit of parts. It may contain technical instructions with information about the quantity.

Preferably, the kit according to the present invention comprises a self-transcribed RNA/DNA system, preferably in lyophilized form, and a suitable vector for reconstitution of the RNA/DNA system. The RNA/DNA system is provided in a container, preferably a container in which the RNA/DNA system is resolubilized. Preferably, the container is connectable to a needleless injection device, for example to fill a disposable syringe of the needleless injection device. The present invention also relates to enhancing (localized) expression of an RNA/DNA system-encoded peptide or protein in skin (mammalian) or muscle, comprising administering an RNA/DNA system as defined herein It's about how to do it.

The invention also provides a method for treating or preventing a disease or disorder comprising administering to a subject in need thereof, in particular a human patient, an RNA/DNA system as defined herein.

The method for treating or preventing a disorder according to the present invention is preferably a vaccination method and/or a gene therapy method as described above.

The composition of the present invention and the self-transcribed RNA/DNA system of the present invention comprising the composition enhances the expression of mRNAi through continuous DdRp amplification or self-transcription and/or 5' cap structure addition in the cytoplasm, and selects it for cancer tissue As it can be delivered for a long period of time, it can be used as a therapeutic target for diseases requiring long-term mRNAi expression while reducing the number of administrations.

BRIEF DESCRIPTION OF THE DRAWINGS The drawings shown below are exemplary only and illustrate the present invention in another way. These drawings should not be construed as limiting the present invention.
1 shows the design of promoter and template DNA sequences for in vitro transcription using T7 RNAP, wherein the double-stranded portion of the promoter is numbered relative to the transcription start site (+1);
Figure 2 is a diagram comparing the promoter nucleotide sequence to which DdRp encoded by the bacteriophage T7, T3, SP6 and K11 binds,
3 is a diagram illustrating a genome editing RNA/DNA system that performs nuclear-independent self-transcription in the cytoplasm;
Figure 4 is a sequence logo showing the most conserved bases around the start codon of more than 10,000 human mRNAs. Large letters indicate higher frequencies, and larger sizes of A and G at position 8 (-3, Kozak position) and Kozak There is a G at the 14th position corresponding to the (+4) position of the sequence,
Figure 5 is <T7 promoter - 5'UTR - T7Pol - muag - A64 - C30 - histone-SL> after cloning the PCR product into the pUC19 vector, by separating the plasmid from the selected strain and processing EcoRI / BamHI restriction enzyme As a result of electrophoresis,
6 is a result of in vitro transcription of the PCR product of the prepared SM_DdRp pUC19 vector having a T7 promoter-T7 polymerase sequence as a template as a template;
7 shows the prepared capped SM_T7pol mRNA (Native RNA), the capped CM/SM_T7pol mRNA (modified RNA), and the capped CM/SM_T7pol mRNA (modified RNA-choliterol) with choliterol added to the 3' end of the prepared RNA in a cell culture medium. It is the result of confirming in vitro stability by RT-PCR after treatment for various times,
Figure 8 is the result of electrophoresis after cloning the PCR product containing the SM_DdRp DNA fragment containing T7 T7RNAP into the pCI-neo vector, separating the plasmid from the selected strain and treating with BglII / HpaI restriction enzyme;
Figure 9 is the result of electrophoresis after cloning the PCR product containing the SM_DdRp DNA fragment containing NP868R-T7RNAP into the pCI-neo vector, separating the plasmid from the selected strain, and treating with BglII / HpaI restriction enzyme;
10 is a DNAi expression cassette clone as an image obtained by cloning a DNAi fragment encoding an mRNA of interest into a pCI-neo vector, digesting a recombinant pCI-neo vector isolated from a selected strain with BglII/HpaI, and gel electrophoresis. is the result of selecting
11 is an image obtained by cloning the PCR amplification product of auto_DdRp and DNAi fragment into pShuttle vector, cutting the plasmid in the selected clone with XhoI/SalI and performing gel electrophoresis, and is the result of selecting auto_DdRp or DNAi expression cassette clone;
12 is a result of co-transformation of a linearized recombinant auto_DdRp or DNAi expression cassette with pAdEasy-1, a supercoil virus DNA plasmid, into BJ5183 bacteria, isolating the recombinant plasmid from the selected strain, treating it with Pac I, and electrophoresis;
13 is an image of the PCR amplification products of auto_DdRp and DNAi fragments obtained by cloning the pSMPUW vector, cutting the plasmid with XhoI/SalI in the selected clone and performing gel electrophoresis, and the result of selecting the auto_DdRp or DNAi expression cassette clone ego,
Figure 14 is an image of auto_(DdRp+EPO) fragment cloned into a binary vector (Ti plAsmid), separated from the selected strain, cut with EcoRI/SalI, and gel electrophoresed, auto_(DdRp+EPO) It is the result of selecting the Ti expression cassette clone,
15 is an image obtained by cloning the mRNA of interest in the pCI neo vector as a control, then separating the plasmid from the selected strain, digesting it with a restriction enzyme, and electrophoresis;
Figure 16 is the result of analyzing the expression of T7 polymerase mRNA in DdRp of auto_DdRp transformed cells using NP868R-T7RNAP. In the control pCI-neo_RSV-F@LS, there is no expression of T7 polymerase mRNA, and DdRp RNA/RSV-F In DNAi@LS transformed cells, it was confirmed that the transfected 5'Cap structure and stabilized DdRp RNA was slowly degraded in the cell, and DdRp RNA/auto_DdRp/RSV-F DNAi@LS DdRp RNA/auto_(DdRp/ RSV-F DNAi)@LS; In cells transformed with the auto(+) DdRp expression system including the auto_(DdRp/RSV-F DNAi) adeno expression system or the auto_(DdRp/RSV-F DNAi) lenti expression system, the first It was confirmed that T7 polymerase binds to the T7 promoter of the auto_DdRp expression cassette and stably synthesizes DdRp through the action of the self-transcription loop synthesizing DdRp in the downstream region.
17 is a result of analyzing the expression of T7 polymerase in DdRp of auto_DdRp transformed cells using NP868R-T7RNAP. As a result, DdRp RNA/RSV-F DNAi@LS auto(-) DdRp expression system and DdRp RNA/ auto_DdRp/RSV-F DNAi@LS DdRp RNA/auto_(DdRp/RSV-F DNAi)@LS; T7 polymerase synthesized in cells transformed with an auto_(DdRp/RSV-F DNAi) adeno expression system or an auto(+) DdRp expression system including an auto_(DdRp/RSV-F DNAi) lenti expression system is at least in the cytoplasm. It is maintained at a high concentration for more than 72 hours,
Figure 18 shows the DdRp RNA/RSV-F DNAi@LS DdRp RNA/auto_DdRp/RSV-F DNAi@LS DdRp RNA/auto_(DdRp/RSV-F DNAi)@ LS; Cells treated with the auto_(DdRp/RSV-F DNAi) adeno expression system or the auto_(DdRp/RSV-F DNAi) lenti expression system observed an increase in RSV-F mRNAi for 24 hours, and at 48 hours, high concentrations for at least 9 days. RSV-F mRNAi could be observed, but in cells treated with control pCI-neo_RSV-F @LS, mRNAi concentrations were similar to those of cells treated with auto_mRNAi@LS, the experimental group, for 24 hours, and 72 hours. From then on, only a slightly lower concentration of RSV-F mRNAi could be measured up to 72 hours,
FIG. 19 shows that DdRp RNA/RSV-F DNAi@LS, auto(-)_mRNAi@LS, is repeatedly synthesized from stabilized capped CM/SM DdRp mRNA in the cytoplasm of 293 T cells transfected with T7 polymerase at high concentrations for a long period of time. By binding to the T7 promoter of the DdRp RNA expression cassette maintained and repeatedly, RNA expression can be maintained continuously for a long period of time, thereby stably and continuously providing the RSV-F protein,
Figure 20 shows the results obtained by separating the total protein at 0, 24, 48 and 72 hours after treatment with the transformant 293T cells by the control group and the unconstitutional group, and the EPO level was determined by ELISA.
21 is a multi_KRASi consisting of <KRAS (G12D) 25 mer ^ 3 - P2A - KRAS (G12V) 25 mer ^ 3 - P2A - KRAS (G13D) 25 mer ^ 3 - P2A - KRAS (G12C) 25 mer ^ 3> KRAS (G12D) 25-mer ^3, KRAS (G12V) 25-mer ^3, -KRAS (G13D) 25-mer ^3 and KRAS (G12C) 25-mer, expressed as a precursor and cleaved in the P2A domain by subsequent operation in 293T cells ^3 is the result of confirming the formation,
Figure 22 shows the test group RNA/DNA1_multi_KRASi@LS, RNA/DNA2_multi_KRASi@LS or RNA/DNA3_multi_KRASi@LS and the capped CM_DdRp mRNA-containing RNA (DdRp RNA) and liposome complex DdRp RNA@LS in 293T cells transformed with Western blot. by ting. Inactivation of Ras activity is a result of confirming the loss of phospho-ERK, which is a downstream ERK activity,
23 shows the protective effect of RNA/DNA1_multi_KRASi@LS, RNA/DNA2_multi_KRASi@LS and RNA/DNA3_multi_KRASi@LS nanoparticles against tumorigenesis after administration of two different CT26 cell doses in an immune tumor model (a) immunization and tumor This is a flow chart of inoculation and (b) immunized mice were prepared by administering nanoparticles 3 times with an interval of 10 days, subcutaneously injecting two different doses of CT26 cells into the immunized mice, and one week after administering the third nanoparticles, female BalB/ c mice (n = 5-8) were administered 3x10 ⁵ CT26 cells/mouse in the right flank, and tumor volume was determined in individual mice and calculated for each group, data means ± SD, Student's t - The assay was performed and the mice were euthanized on the 23rd day after tumor inoculation, (c) the mice administered with the nanoparticles of the present invention were inoculated with CT26 cells (1x10 ⁵ cells/mouse), the tumor was palpable, and the tumor volume (mm ³ ) Tumor growth was monitored every 2 to 3 days after presented as , and data mean ± SD,
Figure 24 shows the therapeutic efficacy of RNA/DNA1_multi_KRASi@LS, RNA/DNA2_multi_KRASi@LS and RNA/DNA3_multi_KRASi@LS nanoparticle inoculation in CT26 tumor model. (a) time course of tumor administration and nanoparticle treatment, (b) 6- After subcutaneous injection of CT26 cells (1x10 ⁵ cells/mouse) into the right flank of 8-week-old female BalB/c, mice (n = 5-8) were randomly divided 2 days after cell injection, and nanoparticles or PBS were administered to each The mice in the group were administered 3 times with an interval of 1 week. Tumors were monitored every 2-3 days and measured using Vernier calipers. Tumor growth curves were plotted by measuring tumor volume over time in each group, and data were expressed as mean ± SD.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples only.

Example 1. Design and manufacture of self-transcribed RNA/DNA systems

The RNA/DNA system capable of continuous mRNAi cytoplasmic expression of the present invention proposes an RNA/DNA system capable of stable expression of target mRNA by DdRp capable of self-transcription. A type of DdRp composed of a single subunit in DdRp in <Table 1>, T7 RNA polymerase (Gene ID: 1261050), T3 RNA polymerase (Gene ID: 14675519) and SP6 RNA polymerase (Gene ID: 19685893) In particular, a system in which the expression cassette is composed of a linear nucleic acid fragment, a circular plasmid virus gene carrier, or a combination thereof that can stably express target mRNA by DdRp using It was prepared as follows. Promoter nucleotide sequences corresponding to these are the same as T7 promoter SEQ ID NO: 1, T3 promoter SEQ ID NO: 2, and SP6 promoter SEQ ID NO: 3 of <FIG. 2>.

An example of the self-transcribed RNA/DNA system of the present invention shown in <Fig. 3> is the self-transcribed RNA/DNA system 1 consisting of a DdRp RNA expression cassette and a DNAi expression cassette containing CM_DdRp mRNA capped in the present invention; self-transcribed RNA/DNA system 2 consisting of a DdRp RNA expression cassette comprising capped CM_DdRp mRNA, an auto_DdRp expression cassette and a DNAi expression cassette; There may be a self-transcribed RNA/DNA system 3 consisting of a DdRp RNA expression cassette comprising capped CM_DdRp mRNA and an auto_(DdRp+DNAi) expression cassette. In the self-transcribed RNA/DNA system of the present invention, the RNA expression cassette is an independently isolated RNA molecule, and the DNA expression cassette may use a plasmid or a viral system.

In the present invention, when the DNA expression cassette is a plasmid, the former self-transcription RNA/DNA system 1 and the liposome delivery system were called auto(-)_DNAi@LS, and the latter two and the liposome delivery system were called auto_DNAi@LS. Auto(-)_DNAi@LS, a complex of the self-transcribed RNA/DNA system 1 of the present invention and liposomes, is a T7 polymerase repeatedly synthesized from a DdRp RNA expression cassette containing a stabilized capped CM_DdRp mRNA. By maintaining and repetitive binding to the T7 promoter of the DdRp RNA expression cassette, target mRNA expression can be maintained stably for a long period of time.

The DdRp self-transcription loop system composed of the self-transcribed RNA/DNA system 2 and the self-transcribed RNA/DNA system 3 of the present invention was referred to as "pT7/T7 polymerase system".

Auto_DNAi@LS, which is a complex of self-transcribed RNA/DNA system 2 or self-transcribed RNA/DNA system 3 and liposomes of the present invention, is initially synthesized from a DdRp RNA expression cassette containing a capped DdRp mRNA stabilized in T7 polymerase. By binding to the T7 promoter of the auto_DdRp expression cassette or the auto_(DdRp+DNAi) expression cassette, T7 polymerase is maintained at a high concentration for a long time in the cytoplasm as a result of autotranscription, and the T7 polymerase is repeatedly expressed by binding to the T7 promoter of the DNAi expression cassette. RNA expression can remain stable for a long time.

In the case of using a viral system for the DNA expression cassette in the self-transcription RNA/DNA system of the present invention, the viral expression cassette encoding the DdRp-binding promoter and the expression RNA and the DdRp RNA expression cassette can be divided and produced, and can be independently transfected. . First, the lentivirus expression cassette is injected, stable transformed cells can be selected, and the DdRp RNA expression cassette can be transfected. Alternatively, the adenovirus expression cassette may be injected and the DdRp RNA expression cassette may be transfected.

Even when the self-transcribed RNA/DNA system of the present invention uses a viral expression cassette, the first T7 polymerase synthesized auto_DdRp expression cassette or auto_(DdRp+ DNAi) By binding to the T7 promoter of the expression cassette, T7 polymerase is maintained at a high concentration for a long time in the cytoplasm as a result of autotranscription, and the T7 polymerase repeatedly binds to the T7 promoter of the DNAi expression cassette, thereby maintaining stable target mRNA expression for a long time. .

1-1. Preparation of DdRp RNA Expression Cassettes

In order to prepare the DdRp RNA expression cassette of the present invention, a DdRp DNA having a T7 promoter capable of producing a T7 polymerase ORF was designed and manufactured, and the DdRp DNA having a T7 promoter obtained from the recombinant pUC19 vector was recombined into a pUC19 cloning vector. After in vitro transcription with a template, a 5'Cap structure was added.

<DdRp DNA Design and Manufacturing>

The DdRp DNA of the present invention is a DNA fragment capable of efficiently synthesizing DdRp mRNA, and a DNA fragment composed of <T7 promoter followed by 5'UTR, DdRp-encoding DNA fragment, 3'UTR> was designed.

In the SM_DdRp DNA of the present invention, the T7 polymerase gene is located downstream of the T7 promoter and 5'UTR in the pUC19 vector, and at the same time muag (mutated alpha-globin-3'UTR), A64 poly(A) sequence, poly(C) sequence (C30) and histone stem-loop sequence (histone-SL) upstream of the sequence. The SM_DdRp DNA structure may be <T7 promoter - 5'UTR - T7Pol - muag - A64 - C30 - histone-SL>.

Preferably, nuclear localization signals (NLS) may be located in the DdRp. DdRp without NLS mostly remains in the cytoplasm, whereas protein containing NLS is localized in the nucleus. DdRp without NLS can be cytoplasmic or hybrid DdRp with NLS can be enzymatically activated in the nucleus to selectively start transcription at the T7 promoter placed on chromosomal or plasmid DNA and stop at specific 3'UTRs.

The base sequence of the unit elements constituting the SM_DdRp DNA fragment is as follows.

The 5*?**?*UTR includes the Kozak consensus.

Kozak consensus sequence ACCAUGG (SEQ ID NO: 31).

Figure 4 is a sequence logo showing the most conserved bases around the start codon of more than 10,000 human mRNAs. Large letters indicate a higher frequency, with a larger size of A and G at position 8 (-3, Kozak position) and a G at position 14 corresponding to position (+4) in the Kozak sequence.

For the purposes of the present invention, NLS (SV40 NLS, PKKKRKVEDPYC) may be added to the N and C-terminus of Bacteriophage T7 RNA Polymerase (T7Pol). The N-terminal addition placed the NLS sequence after the start codon AUG, and the C-terminal addition placed the NLS sequence before the stop codon UAA.

SV40 NLS sequence: 5′-CCG AAG AAG AAG CGA AAG GTC-3′ (SEQ ID NO: 32),

The nucleotide sequence of the ORF of Bacteriophage T7 RNA Polymerase (T7Pol), a type of DdRP selected in the present invention, is as shown in SEQ ID NO: 33.

Bacteriophage T7 RNA Polymerase (T7Pol):

ATGAACACGA TTAACATCGC TAAGAACGAC TTCTCTGACA TCGAACTGGC TGCTATCCCG 60

TTCAACACTC TGGCTGACCA TTACGGTGAG CGTTTAGCTC GCGAACAGTT GGCCCTTGAG 120

CATGAGTCTT ACGAGATGGG TGAAGCACGC TTCCGCAAGA TGTTTGAGCG TCAACTTAAA 180

GCTGGTGAGG TTGCGGATAA CGCTGCCGCC AAGCCTCTCA TCACTACCCT ACTCCCTAAG 240

ATGATTGCAC GCATCAACGA CTGGTTTGAG GAAGTGAAAG CTAAGCGCGG CAAGCGCCCGA 300

CAGCCTTCCA GTTCCTGCAA GAAATCAAGC CGGAAGCCGT AGCGTACAT CACCATTAAG 360

ACCACTCTGG CTTGCCTAAC CAGTGCTGAC AATACAACCG TTCAGGCTGT AGCAAGCGCA 420

ATCGGTCGGG CCATTGAGGA CGAGGCTCGC TTCGGTCGTA TCCGTGACCT TGAAGCTAAG 480

CACTTCAAGA AAAACGTTGA GGAACAACTC AACAAGCGCG TAGGGCACGT CTACAAGAAA 540

GCATTTATGC AAGTTGTCGA GGCTGACATG CTCTCTAAGG GTCTACTCGG TGGCGAGGCG 600

TGGTCTTCGT GGCATAAGGA AGACTCTATT CATGTAGGAG TACGCTGCAT CGAGATGCTC 660

ATTGAGTCAA CCGGAATGGT TAGCTTACAC CGCCAAAATG CTGGCGTAGT AGGTCAAGAC 720

TCTGAGACTA TCGAACTCGC ACCTGATACG CTGAGGCTA TCGCAACCCG TGCAGGTGCG 780

CTGGCTGGCA TCTCTCCGAT GTTCCAACCT TGCGTAGTTC CTCCTAAGCC GTGGACTGGC 840

ATTACTGGTG GTGGCTATTG GGCTAACGGT CGTCGTCCTC TGGCGCTGGT GCGTACTCAC 900

AGTAAGAAAG CACTGATGCG CTACGAGACG TTTACATGC CTGAGGTGTA CAAAGCGATT 960

AACATTGCGC AAAACACCGC ATGGAAAATC AACAAGAAAG TCCTAGCGGT CGCCAACGTA 1020

ATCACCAAGT GGAAGCATTG TCCGGTCGAG GACATCCCTG CGATTGAGCG TGAAGAACTC 1080

CCGATGAAAC CGGAAGACAT CGACATGAAT CCTGAGGCTC TCACCGCGTG GAAACGTGCT 1140

GCCGCTGCTG TGTACCGCAA GGACAAGGCT CGCAAGTCTC GCCGTATCAG CCTTGAGTTC 1200

ATGCTTGAGC AAGCCAATAA GTTTGCTAAC CATAAGGCCA TCTGGTTCCC TTACAACATG 1260

GACTGGCGCG GTCGTGTTTA CGCTGTGTCA ATGTTCAACC CGCAAGGTAA CGATATGACC 1320

AAAGGACTGC TTACGCTGGC GAAAGGTAAA CCAATCGGTA AGGAAGGTTA CTACTGGCTG 1380

AAAATCCACG GTGCAAACTG TGCGGGTGTC GATAAGGTTC CGTTCCCTGA GCGCATCAAG 1420

TTCATTGAGG AAAACCACGA GAACATCATG GCTTGCGCTA AGTCTCCACT GGAGAACACT 1480

TGGTGGGCTG AGCAAGATTC TCCGTTCTGC TTCCTTGCGT TCTGCTTTGA GTACGCTGGG 1540

GTACAGCACC ACGGCCTGAG CTATAACTGC TCCCTTCCGC TGGCGTTTGA CGGGTCTTGC 1620

TCTGGCATCC AGCACTTCTC CGCGATGCTC CGAGATGAGG TAGGTGGTCG CGCGGTTAAC 1680

TTGCTTCCTA GTGAAACCGT TCAGGACATC TACGGGATTG TTGCTAAGAA AGTCAACGAG 1740

ATTCTACAAG CAGACGCAAT CAATGGGACC GATAACGAAG TAGTTACCGT GACCGATGAG 1820

AACACTGGTG AAATCTCTGA GAAAGTCAAG CTGGGCACTA AGGCACTGGC TGGTCAATGG 1880

CTGGCTTACG GTGTTACTCG CAGTGTGACT AAGCGTTCAG TCATGACGCT GGCTTACGGG 1940

TCCAAAGAGT TCGGCTTCCG TCAACAAGTG CTGGAAGATA CCATTCAGCC AGCTATTGAT 2000

TCCGGCAAGG GTCTGATGTT CACTCAGCCG AATCAGGCTG CTGGATACAT GGCTAAGCTG 2060

ATTTGGGAAT CTGTGAGCGT GACGGTGGTA GCTGCGGTTG AAGCAATGAA CTGGCTTAAG 2120

TCTGCTGCTA AGCTGCTGGC TGCTGAGGTC AAAGATAAGA AGACTGGAGA GATTCTTCGC 2180

AACGGTTGCG CTGTGCATTG GGTAACTCCT GATGGTTTCC CTGTGTGGCA GGAATACAAG 2240

AAGCCTATTC AGACGCGCTT GAACCTGATG TTCCTCGGTC AGTTCCGCTT ACAGCCTACC 2300

ATTAACACCA ACAAAGATAG CGAGATTGAT GCACACAAAC AGGAGTCTGG TATCGCTCCT 2360

AACTTTGTAC ACAGCCAAGA CGGTAGCCAC CTTCGTAAGA CTGTAGTGTG GGCACACGAG 2420

AAGTACGGAA TCGAATCTTT TGCACTGATT CACGACTCCT TCGGTACCAT TCCGGCTGAC 2480

GCTGCGAACC TGTTCAAAGC AGTGCGCGAA ACTATGGTTG ACACATATGA GTCTTGTGAT 2540

GTACTGGCTG ATTTCTACGA CCAGTTCGCT GACCAGTTGC ACGAGTCTCA ATTGGACAAA 2600

ATGCCAGCAC TTCCGGCTAA AGGTAACTTG AACCTCCGTG ACATCTTAGA GTCGGACTTC 2660

GCGTTCGCGT AATAA 2675 (SEQ ID NO: 33)

wherein 3'UTR is the sequence 5'-GCCCG a TGGG CCTCCCAACG GGCCCTCCTC CCCTCCTTGC ACCG-3' (SEQ ID NO: 34; muag; underlined nucleotides are mutated compared to wild-type sequence) (alpha-) globin mutated UTR, poly (A) The tail is 64 adenine nucleotides, the poly(C) sequence is 30 cytosine nucleotides, and the histone stem-loop is an RNA sequence consisting of (5'-CAAAGGCUCU UUUCAGAGCC ACCA-3' SEQ ID NO: 35).

DNA fragment encoding muag - A64 - C30 - histone SL: GCCCGATGGG CCTCCCAACG GGCCCTCCTC CCCTCCTTGC ACCG AGATTA ATAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAATTCATGAGCC TCCCCCCCCCCC SEQ ID NO: 36

A64: GGACTAGTAG ATCTAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAA (SEQ ID NO: 37)

C30: TGCATCCCCC CCCCCCCCCC CCCCCCCCCC CCCCCTCTA G (SEQ ID NO: 38)

In the present invention, the SM_DdRp DNA fragment was synthesized with <T7 promoter - 5'UTR - T7Pol - muag - A64 - C30 - histone-SL> (Biothiis, USA), and the forward primer 5'- GAATTC TAAT ACGACTCACT ATAG GGGAGA was used as a template. It was amplified using CCACAACGGT TTCCCTCTAG AAAGAACC AT GAACACGATT AACA -3' (SEQ ID NO: 39) and reverse primer 5'- GGATCC TGGTG GCTCTAAAA GAGCCTTTGG-3' (SEQ ID NO: 40). Here the target sequence of T7pol is underlined. Both ends of the primers used include EcoRI/BamHI recognition sequences.

The amplified PCR product was digested using EcoRI/BamHI enzymes, and then cloning reaction was performed using the pUC19 vector linearized with EcoRI/BamHI enzymes and T4 DNA ligase. The reaction solution was transfected into E. coli, and the plasmid was isolated from the selected strain, treated with EcoRI/BamHI restriction enzymes, and then electrophoresed (FIG. 5).

The recombinant pUC19 prepared above can synthesize RNA by DdRp synthesized from a DdRp RNA expression cassette in prokaryotes and can synthesize DdRp by a protein synthesis tool, so it can be used as a DdRp expression cassette. However, in the present invention, in eukaryotic cells, there is no origin of replication in eukaryotic cells, and thus serves as an expression vector for a short time.

<Preparation of DdRp RNA expression cassette>

In order to start self-transcription of T7 polymerase in the RNA/DNA system of the present invention, the first T7 polymerase supply is required. In the present invention, T7pol mRNA containing nucleotides (CM) with capped chemical modifications to solve the problem of low nuclear membrane permeation A DdRp RNA expression cassette comprising a was prepared.

In order to synthesize capped CM_T7pol mRNA, first, the prepared SM_DdRp pUC19 vector having a T7 promoter-T7 polymerase sequence was amplified using a forward primer (SEQ ID NO: 39) and a reverse primer SEQ ID NO: 40 as a template. Here the target sequence of T7pol is underlined. The PCR product was transcribed in vitro as a template (FIG. 6).

CM/SM_T7pol capped using the HighYield T7 ARCA mRNA Synthiis Kit (m5CTP/Ψ-UTP) (Jena BioScience, USA) using the SM_DdRp PCR product with the T7 promoter prepared above as a T7 promoter-T7 polymerase DNA template as a template A DdRp RNA expression cassette containing mRNA was synthesized in vitro. Specifically, a reaction solution containing anti-reverse cap analog [(ARCA) (3′-O-Me-7mG(5′)ppp(5′)G cap analog)] and chemically modified nucleotides (6 mM In vitro transcription was performed in ARCA, 1.5 mM GTP, 7.5 mM 5-Methyl-CTP, 7.5 mM Pseudo-UTP, 7.5 mM ATP). DNA nuclease was added to remove the dsDNA template, and the remaining capped CM/SM_T7pol mRNA was purified by LiCl extraction and ethanol precipitation.

As a control, the synthesis of capped T7pol mRNA in which a 5'cap structure was added to the native RNA transcript in the SM_DdRp vector was performed using the HiScribe ^™ T7 ARCA mRNA Kit (NEB, USA).

7, the prepared capped SM_T7pol mRNA (Native RNA), the capped CM_T7pol mRNA (modified RNA), and the capped CM_T7pol mRNA (modified RNA-choliterol) with cholesterol added to the 3' end were added to the cell culture medium. In vitro stability was confirmed by RT-PCR after processing the prepared RNA for various times. The prepared capped CM_T7pol mRNA had very superior in vitro safety than the capped T7pol mRNA, and the capped CM_T7pol mRNA (modified RNA-choliterol) with choliterol added to the 3' end had the highest stability.

Capped CM_T7pol mRNA can also be added with a native cap (m7G(5')ppp(5')G; m7GpppG) to the IVT product with the ScriptCap m7G capping system (Epicentre Biotechnologii, USA).

1-2. Preparation of auto_DdRp and auto_chimeric DdRp expression cassettes

The auto_DdRp DNA expression cassette and the auto_(DdRp+DNAi) expression cassette of the present invention can be prepared using a plasmid or a viral vector. The auto_DdRp DNA expression cassette and the auto_(DdRp+DNAi) expression cassette are stored in a plasmid or viral vector with an auto_DdRp DNA fragment (T7 promoter - 5'UTR - DdRp DNA (or chimeric NP868R-T7RNAP DNA) - muag - A64 - C30 - histone- SL> was inserted.As shown below, the synthesized DdRp and chimeric DdRp DNA fragments were amplified by PCR as a template to obtain a large amount of DdRp and chimeric DdRp DNA fragments that are easy to clone. It was recombined into the vector.

The expression vectors used in this example were mammalian expression plasmid vector pCI-neo vector (Promega, USA), pShuttle in AdEasy Adenoviral Vector System (Agilent Technologii, USA), ViraSafe™ Lentiviral Exprision System (Cell Biolabs, USA). pSMPUW-IRES-Blasticidin Lentiviral Exprision Vector, shuttle vector pSMPUW, binary vector for plant expression (Ti plasmid), etc., but not limited thereto. In this example, only the plasmid vector was described by limiting it, and the viral vector can be sufficiently prepared with reference to Examples 1-4 below.

<auto_DdRp expression cassette>

In this example, an auto_SM_DdRp expression cassette including a DNA fragment providing DdRp was designed using a plasmid.

The pCI-neo vector (Promega, USA) capable of constructing a stable cell line with a mammalian expression vector was digested with BglII/HpaI restriction enzyme treatment and <T7 promoter followed by 5'UTR, DdRp-encoding DNA fragment, muag, A64 The auto_SM_DdRp expression cassette was prepared by inserting an SM_DdRp DNA fragment consisting of a poly(A) sequence, a poly(C) sequence (C30), and a histone stem-loop sequence (histone-SL) sequence>.

Nuclear localization signals (NLS) may be located in the DdRp. DdRp without NLS mostly remains in the cytoplasm, whereas protein containing NLS is localized in the nucleus. DdRp without NLS can be cytoplasmic or hybrid DdRp with NLS can be enzymatically activated in the nucleus to selectively start transcription at the T7 promoter placed on chromosomal or plasmid DNA and stop at a specific 3*?**?*UTR. there is.

The base sequence of the unit elements constituting the SM_DdRp DNA fragment is as follows.

The SM_DdRp DNA fragment synthesized in Example 1-1 was forward primer 5'- AGATCT TTAA TACGACTCAC TATAG GGGAG ACCACAACGG TTTCCCTCTA GAAAGAACC A TGAACACGAT TAACA -3' (SEQ ID NO: 43) and reverse primer 5'- GTTAAC TGGAGT GCTCTAA-3 ' (SEQ ID NO: 44) was used to amplify. Here the target sequence of T7pol is underlined. Both ends of the primers used include BglII/HpaI recognition sequences.

The PCR product containing the amplified SM_DdRp DNA fragment was digested using BglII/HpaI enzymes, and then subjected to a cloning reaction using a pCI-neo vector linearized with BglII/HpaI enzymes and T4 DNA ligase. The reaction solution was transfected into E. coli, and the plasmid was isolated from the selected strain, treated with BglII/HpaI restriction enzymes, and then electrophoresed (FIG. 8).

In the above <T7 promoter followed by 5'UTR, DNA fragment encoding DdRp, muag, A64 poly(A) sequence, poly(C) sequence (C30) and histone stem-loop sequence (histone-SL) sequence> The recombinant pCI-neo vector constructed from the SM_DdRp DNA fragment was referred to as an auto_DdRp expression cassette.

<auto_chimeric DdRp expression cassette>

The auto_chimeric DdRp expression cassette of the present invention may use a plasmid or a viral vector. In this Example, an auto_chimeric DdRp expression cassette including a DNA fragment providing DdRp was designed using a plasmid.

A pCI-neo vector (Promega, USA) capable of constructing a stable cell line as a mammalian expression vector was digested with BglII/HpaI restriction enzyme treatment and <T7 promoter followed by 5'UTR, a DNA fragment encoding chimeric DdRp, muag; The auto_chimeric DdRp expression cassette was prepared by inserting an SM_DdRp DNA fragment consisting of A64 poly(A) sequence, poly(C) sequence (C30) and histone stem-loop sequence (histone-SL) sequence>.

The DNA fragment encoding the chimeric DdRp of the present invention is the ORF of NP868R-T7RNAP (SEQ ID NO: 21). One plasmid encoding a fusion between NP868R, the mRNA capping enzyme of African swine fever virus, and the wild-type phage DdRp of bacteriophage T7 was synthesized. This capping enzyme was fused to the amino terminus of T7 RNA polymerase by a (Gly3Ser)4 linker.

The nucleotide sequence for the ORF of NP868R-T7RNAP is shown in SEQ ID NO: 21 below.

NP868R-T7RNAP:

ATGGAATTCG CCAGCCTGGA CAACCTGGTG GCCAGATAACC AGCGGTGCTT CAACGACCAG 60

AGCCTGAAGA ACAGCACCAT CGAGCTGGAA ATCCGGTTCC AGCAGATCAA CTTCCTGCTG 120

TTCAAGACCG TGTACGAGGC CCTGGTCGCC CAGGAAATCC CCAGCACCAT CAGCCACAGC 180

ATCCGGTGCA TCAAGAAGGT GCACCACGAG AACCACTGCC GGGAGAAGAT CCTGCCCAGC 240

GAGAACCTGT ACTTCAAGAA ACAGCCCCTG ATGTTCTTCA AGTTCAGCGA GCCCGCCAGC 300

CTGGGCTGTA AAGTGTCCCT GGCCATCGAG CAGCCCATCC GGAAGTTCAT CCTGGACAGC 360

AGCGTGCTGG TCCGGCTGAA GAACCGGACC ACCTTCCGGG TGTCCGAGCT GTGGAAGATC 420

GAGCTGACCA TCGTGAAGCA GCTGATGGGC AGCGAGGTGT CAGCCAAGCT GGCCGCCTTC 480

AAGACCCTGC TGTTCGACAC CCCCGAGCAG CAGACCACCA AGAACATGAT GACCCTGATC 540

AACCCCGACG ACGAGTACCT GTACGAGATC GAGATCGAGT ACACCGGCAA GCCTGAGAGC 600

CTGACAGCCG CCGACGTGAT CAAGATCAAG AACACCGTGC TGACACTGAT CAGCCCCAAC 660

CACCTGATGC TGACCGCCTA CCACCAGGCC ATCGAGTTTA TCGCCAGCCA CATCCTGAGC 720

AGCGAGATCC TGCTGGCCCG GATCAAGAGC GGCAAGTGGG GCCTGAAGAG ACTGCTGCCC 780

CAGGTCAAGT CCATGACCAA GGCCGACTAC ATGAAGTTCT ACCCCCCCGT GGGCTACTAC 840

GTGACCGACA AGGCCGACGG CATCCGGGGC ATTGCCGTGA TCCAGGACAC CCAGATCTAC 900

GTGGTGGCCG ACCAGCTGTA CAGCCTGGGC ACCACCGGCA TCGAGCCCCT GAAGCCCACC 960

ATCCTGGACG GCGAGTTCAT GCCCGAGAAG AAAGAGTTCT ACGGCTTTGA CGTGATCATG 1020

TACGAGGGCA ACCTGCTGAC CCAGCAGGGC TTCGAGACAC GGATCGAGAG CCTGAGCAAG 1080

GGCATCAAGG TGCTGCAGGC CTTCAACATC AAGGCCGAGA TGAAGCCCTT CATCAGCCTG 1140

ACCTCCGCCG ACCCCAACGT GCTGCTGAAG AATTTCGAGA GCATCTTCAA GAAGAAAACC 1200

CGGCCCTACA GCATCGACGG CATCATCCTG GTGGAGCCCG GCAACAGCTA CCTGAACACC 1260

AACACCTTCA AGTGGAAGCC CACCTGGGAC AACACCCTGG ACTTTCTGGT CCGGAAGTGC 1320

CCCGAGTCCC TGAACGTGCC CGAGTACGCC CCCAAGAAGG GCTTCAGCCT GCATCTGCTG 1380

TTCGTGGGCA TCAGCGGCGA GCTGTTTAAG AAGCTGGCCC TGAACTGGTG CCCCGGCTAC 1440

ACCAAGCTGT TCCCCGTGAC CCAGCGGAAC CAGAACTACT TCCCCGTGCA GTTCCAGCCC 1500

AGCGACTTCC CCCTGGCCTT CCTGTACTAC CACCCCGACA CCAGCAGCTT CAGCAACATC 1560

GATGGCAAGG TGCTGGAAAT GCGGTGCCTG AAGCGGGAGA TCAACTACGT GCGCTGGGAG 1620

ATCGTGAAGA TCCGGGAGGA CCGGCAGCAG GATCTGAAAA CCGGCGGCTA CTTCGGCAAC 1680

GACTTCAAGA CCGCCGAGCT GACCTGGCTG AACTACATGG ACCCCTTCAG CTTCGAGGAA 1740

CTGGCCAAGG GACCCAGCGG CATGTACTTC GCTGGCGCCA AGACCGGCAT CTACAGAGCC 1800

CAGACCGCCC TGATCAGCTT CATCAAGCAG GAAATCATCC AGAAGATCAG CCACCAGAGC 1860

TGGGTGATCG ACCTGGGCAT CGGCAAGGGC CAGGACCTGG GCAGATACCT GGACGCCGGC 1920

GTGAGACACC TGGTCGGCAT CGATAAGGAC CAGACAGCCC TGGCCGAGCT GGTGTACCGG 1980

AAGTTCTCCC ACGCCACCAC CAGACAGCAC AAGCACGCCA CCAACATCTA CGTGCTGCAC 2040

CAGGATCTGG CCGAGCCTGC CAAAGAAATC AGCGAGAAAG TGCACCAGAT CTATGGCTTC 2100

CCCAAAGAGG GCGCCAGCAG CATCGTGTCC AACCTGTTCA TCCACTACCT GATGAAGAAC 2160

ACCCAGCAGG TCGAGAACCT GGCTGTGCTG TGCCACAAGC TGCTGCAGCC TGGCGGCATG 2220

GTCTGGTTCA CCACCATGCT GGGCGAACAG GTGCTGGAAC TGCTGCACGA GAACCGGATC 2280

GAACTGAACG AAGTGTGGGA GGCCCGGGAG AACGAGGTGG TCAAGTTCGC CATCAAGCGG 2340

CTGTTCAAAG AGGACATCCT GCAGGAAACC GGCCAGGAAA TCGGCGTCCT GCTGCCCTTC 2400

AGCAACGGCG ACTTCTACAA TGAGTACCTG GTCAACACCG CCTTTCTGAT CAAGATTTTC 2460

AAGCACCATG GCTTTAGCCT CGTGCAGAAG CAGAGCTTCA AGGACTGGAT CCCCGAGTTC 2520

CAGAACTTCA GCAAGAGCCT GTACAAGATC CTGACCGAGG CCGACAAGAC CTGGACCAGC 2580

CTGTTCGGCT TCATCTGCCT GCGGAAGAAC GGGCCCGGCG GAGGCGGAAG TGGAGGCGGA 2640

GGAAGCGGAG GGGGAGGATC TGGCGGCGGA GGCAGCCTCG AGAACACCAT CAATATCGCC 2700

AAGAACGACT TCAGCGACAT CGAGCTGGCC GCCATCCCTT TCAACACCCT GGCCGACCAC 2760

TATGGCGAGC GGCTGGCCAG AGAACAGCTG GCCCTGGAAC ACGAGAGCTA CGAAATGGGC 2820

GAGGCCCGGT TCCGGAAGAT GTTCGAGAGA CAGCTGAAGG CCGGCGAGGT GGCCGATAAT 2880

GCCGCCGCTA AGCCCCTGAT CACCACCCTG CTGCCCAAGA TGATCGCCCG GATCAACGAT 2940

TGGTTCGAGG AAGTGAAGGC CAAGCGGGGC AAGAGGCCCA CCGCCTTCCA GTTTCTGCAG 3000

GAAATCAAGC CCGAGGCCGT GGCCTACATC ACCATCAAGA CCACCCTGGC CTGCCTGACC 3060

AGCGCCGACA ATACCACCGT GCAGGCTGTC GCTTCTGCCA TCGGCAGAGC CATCGAGGAC 3120

GAGGCCAGAT TCGGCAGAAT CCGGGACCTG GAAGCCAAGC ACTTCAAGAA AAACGTGGAG 3180

GAACAGCTGA ACAAGCGCGT GGGCCACGTG TACAAGAAAG CCTTCATGCA GGTGGTGGAG 3240

GCCGACATGC TGAGCAAGGG CCTGCTGGGC GGAGAAGCCT GGTCCAGCTG GCACAAAGAG 3300

GACAGCATCC ACGTCGGCGT GCGGTGCATC GAGATGCTGA TCGAGTCGAC CGGCATGGTG 3360

TCCCTGCACA GGCAGAATGC CGGCGTGGTG GGCCAGGACA GCGAGACAAT CGAACTGGCC 3420

CCCGAGTATG CCGAGGCCAT TGCCACAAGA GCCGGCGCTC TGGCCGGCAT CAGCCCCATG 3480

TTCCAGCCCT GCGTGGTGCC TCCTAAGCCC TGGACAGGCA TCACAGGCGG CGGATACTGG 3540

GCCAACGGCA GACGCCCTCT GGCTCTGGTG CGGACCCACA GCAAGAAAGC CCTGATGCGC 3600

TACGAGGACG TGTACATGCC CGAGGTGTAC AAGGCCATCA ACATTGCCCA GAACACCGCC 3660

TGGAAGATCA ACAAGAAAGT GCTGGCCGTG GCCAATGTGA TCACCAAGTG GAAGCACTGC 3720

CCCGTGGAGG ACATCCCCGC CATCGAGCGG GAGGAACTGC CCATGAAGCC CGAGGACATC 3780

GACATGAACC CCGAGGCCCT GACAGCCTGG AAAAGGGCCG CTGCCGCCGT GTACCGGAAG 3840

GACAAGGCCC GGAAGTCCCG GCGGATCAGC CTGGAGTTCA TGCTGGAACA GGCCAACAAG 3900

TTCGCCAACC ACAAGGCCAT TTGGTTCCCT TACAACATGG ACTGGCGGGG CAGAGTGTAC 3960

GCCGTGAGCA TGTTCAATCC ACAAGGCAAC GACATGACCA AGGGGCTGCT GACCCTGGCC 4020

AAGGGCAAGC CCATCGGCAA AGAGGGCTAC TACTGGCTGA AGATCCACGG CGCCAACTGC 4080

GCTGGCGTGG ACAAGGTGCC CTTCCCAGAG CGGATCAAGT TCATCGAGGA AAACCACGAG 4140

AACATCATGG CCTGCGCCAA GAGCCCTCTA GAAAACACTT GGTGGGCCGA GCAGGACAGC 4200

CCCTTCTGCT TCCTGGCCTT CTGCTTTGAG TACGCCGGAG TGCAGCACCA CGGCCTGAGC 4260

TACAACTGCA GCCTGCCCCT GGCCTTCGAT GGCAGCTGCA GCGGCATCCA GCACTTCAGC 4320

GCCATGCTGA GGGACGAAGT GGGCGGCAGA GCCGTGAATC TGCTGCCAAG CGAGACAGTG 4380

CAGGACATCT ACGGGATCGT GGCCAAGAAA GTGAACGAGA TCCTGCAGGC CGACGCCATC 4440

AACGGCACCG ACAACGAGGT GGTGACCGTG ACCGATGAGA ACACCGGCGA GATCAGCGAG 4500

AAAGTGAAGC TCGGCACCAA GGCCCTGGCT GGCCAGTGGC TGGCCTACGG CGTGACCAGA 4560

TCCGTGACCA AGCGGAGCGT GATGACACTG GCCTATGGCA GCAAAGAGTT CGGCTTCCGG 4620

CAGCAGGTGC TGGAAGATAC CATCCAGCCT GCCATCGACA GCGGCAAGGG GCTGATGTTC 4680

ACCCAGCCCA ACCAGGCCGC TGGCTACATG GCCAAGCTCA TATGGGAGAG CGTGTCCGTG 4740

ACAGTGGTGG CCGCCGTGGA GGCCATGAAT TGGCTGAAGT CCGCCGCCAA ACTGCTGGCC 4800

GCTGAAGTGA AGGACAAAAA GACCGGCGAA ATCCTGCGGA AGAGATGCGC CGTGCACTGG 4860

GTGACCCCCG ATGGCTTCCC CGTGTGGCAG GAGTACAAGA AGCCCATCCA GACCCGGCTG 4920

AACCTGATGT TCCTGGGCCA GTTCAGACTG CAGCCCACCA TCAACACCAA CAAGGACTCC 4980

GAGATCGACG CCCACAAGCA GGAAAGCGGC ATTGCCCCCA ACTTCGTGCA CAGCCAGGAC 5040

GGCAGCCACC TGAGAAAGAC CGTGGTGTGG GCTCACGAGA AGTACGGCAT CGAGAGCTTC 5100

GCCCTGATCC ACGACAGCTT CGGCACCATT CCCGCCGACG CCGCCAACCT GTTCAAGGCC 5160

GTGCGGGAGA CAATGGTGGA CACCTACGAG AGCTGCGACG TGCTGGCCGA CTTCTACGAC 5220

CAGTTCGCCG ACCAGCTGCA CGAGAGCCAG CTGGACAAGA TGCCCGCCCT GCCCGCCAAG 5280

GGGAACCTGA ACCTGCGGGA CATCCTGGAA AGCGACTTCG CCTTCGCCTG A 5331 (SEQ ID NO: 45)

Nuclear localization signals (NLS) may be located in the chimeric DdRp. The chimeric DdRp without NLS mostly remains in the cytoplasm, whereas the protein containing NLS is localized in the nucleus. Chimeric DdRp without NLS can be activated enzymatically in the nucleus, either in the cytoplasm or chimeric DdRp with NLS to selectively start transcription at the T7 promoter placed in chromosomal or plasmid DNA and stop at specific T7 terminator sequences.

The base sequence of the unit elements constituting the SM_chimeric DdRp DNA fragment is as follows.

In the present invention, a SM_DdRp DNA fragment consisting of <T7 promoter - 5'UTR - NP868R-T7RNAP - muag - A64 - C30 - histone-SL> was synthesized (Biosynthiis, USA), and the forward primer 5'-AGATCT TAAT ACGACTCACT was used as a template. Amplification was performed using ATAG GAGACC ACAACGGTTTC CCTCTAGAAA GAACC ATGGAATTC GCCAGCCTGG A -3' (SEQ ID NO: 43) and reverse primer 5'-GTTAAC TGGT GGCTCTAAAA GAGCCTTTGG -3' (SEQ ID NO: 44). Here the target sequence of T7pol is underlined. Both ends of the primers used include BglII/HpaI recognition sequences.

The PCR product containing the amplified SM_DdRp DNA fragment was digested using BglII/HpaI enzymes, and then subjected to a cloning reaction using a pCI-neo vector linearized with BglII/HpaI enzymes and T4 DNA ligase. The reaction solution was transfected into E. coli, and the plasmid was isolated from the selected strain, treated with BglII/HpaI restriction enzymes, and then electrophoresed (FIG. 9).

In the above <T7 promoter followed by 5'UTR, DNA fragment encoding NP868R-T7RNAP, 3'UTR, A64 poly(A) sequence, poly(C) sequence (C30) and histone stem-loop sequence (histone-SL) The recombinant pCI-neo vector constructed with the SM_DdRp DNA fragment consisting of sequence> was referred to as an auto_SM_chimeric DdRp expression cassette.

1-3. Preparation of DNAi Expression Cassettes

The expression vectors used in this example were mammalian expression plasmid vector pCI-neo vector (Promega, USA), pShuttle in AdEasy Adenoviral Vector System (Agilent Technologii, USA), ViraSafe™ Lentiviral Exprision System (Cell Biolabs, USA). pSMPUW-IRES-Blasticidin Lentiviral Exprision Vector, shuttle vector pSMPUW, binary vector for plant expression (Ti plasmid), etc., but not limited thereto. In this example, only the plasmid vector was described by limiting it, and the viral vector can be sufficiently prepared with reference to Examples 1-4 below.

In this example, a DNAi expression cassette containing DNAi providing mRNAi was designed using a plasmid. The pCI-neo vector (Promega, USA) capable of constructing a stable cell line as a mammalian expression vector was digested with BglII/HpaI restriction enzyme treatment and <T7 promoter followed by 5'UTR, DNAi encoding the selected mRNAi and 3' The DNAi expression cassette was prepared by inserting a DNAi fragment consisting of UTR>.

The constructed SM_DNAi fragment was synthesized by chemical synthesis (Biosynthiis, USA) and amplified again by PCR as a template. At this time, BglII/HpaI recognition sequences were given to both ends using PCR primers. The amplified PCR product was inserted into a pCI-neo vector (Promega, USA) using T4 DNA ligase after treatment with BglII/HpaI restriction enzymes. The resulting DNAi expression cassette was confirmed by DNA sequencing.

The DNAi fragment designed below was prepared by organic synthesis (Biosynthesis, USA). Here, <muag - A64 - C30 - histone SL> was used for 3*?**?*UTR. It was inserted into the pCI-neo vector (Promega, USA), AdEasy Adenoviral Vector System (Agilent Technologii, USA) and ViraSafe™ Lentiviral Exprision System (Cell Biolabs, USA), which can construct a stable cell line with a mammalian expression vector.

The synthesized DNAi fragment was amplified using a forward primer 5'- AGATCT TAAT ACGACTCACT ATAG GGAGACCAC AACGGUUUCC CUCU -3' (SEQ ID NO: 43) and a reverse primer 5'- GTTAAC TGGT GGCTCTAAAA GAGCCTTTGG -3' (SEQ ID NO: 44). Here the target sequence of T7pol is underlined. Both ends of the primers used include BglII/HpaI recognition sequences.

Specifically, 5 μl 10×MgCl2 reaction buffer, 1 μl forward primer (20 μM), 1 μl reverse primer (20 μM), 1 μl dNTP (10mM), 1 μl prepared DNA fragment (10ng/μl), 0.5 μl enzyme PCR was performed by adding 40.5 μl dH2O to the reaction solution containing the mix (ie, polymerase).

The amplification product was treated with BglII/HpaI restriction enzymes, mixed with a pCI-neo vector linearized with BglII/HpaI restriction enzymes, and DNA ligase was added to perform a cloning reaction. 2-3 μl of the pCI-neo reaction solution was transduced into E. coli, plated on an ampicillin selection plate, and incubated at 37° C. overnight.

Pick 6-12 colonies and inoculate them in 3 ml of LB growth medium containing 50 μg/ml of ampicillin each. Incubate overnight on a shaker at 37°C. Recombinant pCI-neo vectors were isolated using standard procedures or a commercial Qiaprep Miniprep kit (Qiagen, USA). The recombinant pCI-neo vector was digested with BglII/HpaI and gel electrophoresis was performed to select a DNAi expression cassette clone (FIG. 10).

The mRNAi used in this example is the rabies virus G protein (RAV-G), the respiratory syncytial virus (RSV) long strain RSV-F protein, murine EPO or multiple mRNA encoding the cancer antigen (KRASi) of the oncogene KRAS mutant of

The construction sequence of mRNAi and SM_mRNAi used in the present invention is as follows.

G protein of rabies virus (RAV-G)

For the preparation of the DNAi expression cassette of the present invention, the following vector encoding the rabies virus G protein (RAV-G) was prepared:

5'UTR-RAV-G(GC)-muag-A64-C30-histoneSL (R2403) downstream of the T7 promoter of the pCI-neo vector is a GC-rich sequence encoding the G protein of the rabies virus after the T7 promoter, mutated 3'UTR (muag) of alpha globin, A64 poly(A) sequence, poly(C) sequence (C30) and histone stem-loop sequence (histone-SL):

RAV-G(GC) - muag - A64 - C30 - Histone SL

GGGAGACCAC AACGGUUUCC CUCUAGAAAG AACC AUGGU GCCCCAGGCC CUGCUCUUCG 60

UCCCGCUGCU GGUGUUCCCC CUCUGCUUCG GCAAGUUCCC CAUCUACACC AUCCCCGACA 120

AGCUGGGGCC GUGGAGCCCC AUCGACAUCC ACCACCUGUC CUGCCCCAAC AACCUCGUGG 180

UCGAGGACGA GGGCUGCACC AACCUGAGCG GGUUCUCCUA CAUGGAGCUG AAGGUGGGCU 240

ACAUCAGCGC CAUCAAGAUG AACGGGUUCA CGUGCACCGG CGUGGUCACC GAGGCGGAGA 300

CCUACACGAA CUUCGUGGGC UACGUGACCA CCACCUUCAA GCGGAAGCAC UUCCGCCCCA 360

CGCCGGACGC CUGCCGGGCC GCCUACAACU GGAAGAUGGC CGGGGACCCC CGCUACGAGG 420

AGUCCCUCCA CAACCCCUAC CCCGACUACC ACUGGCUGCG GACCGUCAAG ACCACCAAGG 480

AGAGCCUGGU GAUCAUCUCC CCGAGCGUGG CGGACCUCGA CCCCUACGAC CGCUCCCUGC 540

ACAGCCGGGU CUUCCCCGGC GGGAACUGCU CCGGCGUGGC CGUGAGCUCC ACGUACUGCA 600

GCACCAACCA CGACUACACC AUCUGGAUGC CCGAGAACCC GCGCCUGGGG AUGUCCUGCG 660

ACAUCUUCAC CAACAGCCGG GGCAAGCGCG CCUCCAAGGG CAGCGAGACG UGCGGGUUCG 720

UCGACGAGCG GGGCCUCUAC AAGUCCCUGA AGGGGGCCUG CAAGCUGAAG CUCUGCGGCG 780

UGCUGGGCCU GCGCCUCAUG GACGGGACCU GGGUGGCGAU GCAGACCAGC AACGAGACCA 840

AGUGGUGCCC CCCCGGCCAG CUGGUCAACC UGCACGACUU CCGGAGCGAC GAGAUCGAGC 900

ACCUCGUGGU GGAGGAGCUG GUCAAGAAGC GCGAGGAGUG CCUGGACGCC CUCGAGUCCA 960

UCAUGACGAC CAAGAGCGUG UCCUUCCGGC GCCUGAGCCA CCUGCGGAAG CUCGUGCCCG 1020

GGUUCGGCAA GGCCUACACC AUCUUCAACA AGACCCUGAU GGAGGCCGAC GCCCACUACA 1080

AGUCCGUCCG CACGUGGAAC GAGAUCAUCC CGAGCAAGGG GUGCCUGCGG GUGGGCGGCC 1140

GCUGCCACCC CCACGUCAAC GGGGUGUUCU UCAACGGCAU CAUCCUCGGG CCCGACGGCA 1200

ACGUGCUGAU CCCCGAGAUG CAGUCCAGCC UGCUCCAGCA GCACAUGGAG CUGCUGGUCU 1260

CCAGCGUGAU CCCGCUCAUG CACCCCCUGG CGGACCCCUC CACCGUGUUC AAGAACGGGG 1320

ACGAGGCCGA GGACUUCGUC GAGGUGCACC UGCCCGACGU GCACGAGCGG AUCAGCGGCG 1380

UCGACCUCGG CCUGCCGAAC UGGGGGAAGU ACGUGCUGCU CUCCGCCGGC GCCCUGACCG 1440

CCCUGAUGCU GAUCAUCUUC CUCAUGACCU GCUGGCGCCG GGUGAACCGG AGCGAGCCCA 1500

CGCAGCACAA CCUGCGCGGG ACCGGCCGGG AGGUCUCCGU GACCCCGCAG AGCGGGAAGA 1560

UCAUCUCCAG CUGGGAGUCC UACAAGAGCG GCGGCGAGAC CGGGCUGUGA GGACUAGUUA 1620

UAAGACUGAC UAGCCCGAUG GGCCUCCCAA CGGGCCCUCC UCCCCUCCUU GCACCGAGAU 1680

UAAUAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1740

AAAAAAAAUG CAUCCCCCCC CCCCCCCCCC CCCCCCCCCC CCCCAAAGGC UCUUUUCAGA 1820

GCCACCAGAA UU 1832 (SEQ ID NO: 24)

RSV-F protein of RSV long strain

For the preparation of the DNAi expression cassette of the present invention, the following vector encoding the RSV-F protein of a respiratory syncytial virus (RSV) long strain was prepared.

Fusion protein F (amino acid sequence according to SEQ ID NO: 26) of RSV strain ATCC VR-26 long:

MELPILKANA ITTILAAVTF CFASSQNITE EFYQSTCSAV SKGYLSalRT GWYTSVITIE LSNIKENKCN GTDAKVKLIN QELDKYKNAV TELQLLMQST TAANNRARRE LPRFMNYTLN NTKKTNVTLS KKRKRRFLGF LLGVGSAIAS GIAVSKVLHL EGEVNKIKSA LLSTNKAVVS LSngVSVLTS KVLDLKNYID KQLLPIVNKQ SCRISNIETV IEFQQKNNRL LEITREFSVN AGVTTPVSTY MLTNSELLSL INDMPITNDQ KKLMSNNVQI VRQQSYSIMS IIKEEVLAYV VQLPLYGVID TPCWKLHTSP LCTTNTKEGS NICLTRTDRG WYCDNAGSVS FFPQAETCKV QSNRVFCDTM NSLTLPSEVN LCNVDIFNPK YDCKIMTSKT DVSSSVITSL GAIVSCYGKT KCTASNKNRG IIKTFSngCD YVSNKGVDTV SVGNTLYYVN KQEGKSLYVK GEPIINFYDP LVFPSDEFDA SISQVNEKIN QSLAFIRKSD ELLHHVNAGK STTNIMITTI IIVIIVILLS LIAVGLLLYC KARSTPVTLS KDQLSGINNI AFSN (SEQ ID NO:25)

According to the first preparation, a DNA sequence encoding the above-mentioned mRNA was prepared. An expression cassette containing DNAi encoding the RNA sequence R2510 (SEQ ID NO: 64) was prepared by introducing 5'TOP-UTR derived from ribosomal protein 32L, and the wild-type coding sequence was modified by introducing a GC-optimal sequence for stabilization and then the 3'UTR (muag), A64 poly(A) sequence, poly(C) sequence (C30) and histone stem-loop sequence (histone-SL) of the mutated alpha globin were added.

The sequence of the corresponding mRNA (SEQ ID NO: 26) is shown.

RSV-F long (GC)

GGGAGACCAC AACGGUUUCC CUCUAGAAAG AACC AUGGAGC UGCCCAUCCU CAAGGCCAAC 60

GCCAUCACCA CCAUCCUGGC GGCCGUGACG UUCUGCUUCG CCAGCUCCCA GAACAUCACC 120

GAGGAGUUCU ACCAGAGCAC CUGCUCCGCC GUCAGCAAGG GCUACCUGUC CGCCCUCCGG 180

ACCGGGUGGU ACACGAGCGU GAUCACCAUC GAGCUGUCCA ACAUCAAGGA GAACAAGUGC 240

AACGGCACCG ACGCGAAGGU GAAGCUGAUC AACCAGGAGC UCGACAAGUA CAAGAACGCC 300

GUCACCGAGC UGCAGCUGCU CAUGCAGAGC ACGACCGCCG CCAACAACCG CGCGCGGCGC 360

GAGCUGCCGC GGUUCAUGAA CUACACCCUG AACAACACCA AGAAGACGAA CGUGACCCUC 420

UCCAAGAAGC GCAAGCGGCG CUUCCUGGGG UUCCUGCUCG GCGUGGGGAG CGCCAUCGCC 480

UCCGGCAUCG CCGUCAGCAA GGUGCUGCAC CUGGAGGGCG AGGUGAACAA GAUCAAGUCC 540

GCCCUCCUGA GCACCAACAA GGCGGUCGUG UCCCUGAGCA ACGGGGUGUC CGUCCUCACC 600

AGCAAGGUGC UGGACCUGAA GAACUACAUC GACAAGCAGC UCCUGCCCAU CGUGAACAAG 660

CAGUCCUGCC GGAUCAGCAA CAUCGAGACG GUCAUCGAGU UCCAGCAGAA GAACAACCGC 720

CUGCUCGAGA UCACCCGGGA GUUCAGCGUG AACGCCGGCG UGACCACCCC CGUCUCCACG 780

UACAUGCUGA CCAACAGCGA GCUGCUCUCC CUGAUCAACG ACAUGCCCAU CACCAACGAC 840

CAGAAGAAGC UGAUGAGCAA CAACGUGCAG AUCGUGCGCC AGCAGUCCUA CAGCAUCAUG 900

UCCAUCAUCA AGGAGGAGGU CCUCGCCUAC GUGGUGCAGC UGCCGCUGUA CGGGGUCAUC 960

GACACCCCCU GCUGGAAGCU CCACACGAGC CCCCUGUGCA CCACCAACAC CAAGGAGGGC 1020

UCCAACAUCU GCCUGACGCG GACCGACCGC GGGUGGUACU GCGACAACGC CGGCAGCGUG 1080

UCCUUCUUCC CCCAGGCCGA GACCUGCAAG GUCCAGAGCA ACCGGGUGUU CUGCGACACC 1140

AUGAACUCCC UCACGCUGCC GAGCGAGGUG AACCUGUGCA ACGUCGACAU CUUCAACCCC 1200

AAGUACGACU GCAAGAUCAU GACCUCCAAG ACCGACGUGA GCUCCAGCGU GAUCACCUCC 1260

CUCGGCGCGA UCGUCAGCUG CUACGGGAAG ACGAAGUGCA CCGCCAGCAA CAAGAACCGC 1320

GGCAUCAUCA AGACCUUCUC CAACGGGUGC GACUACGUGA GCAACAAGGG CGUGGACACC 1380

GUCUCCGUGG GCAACACCCU GUACUACGUG AACAAGCAGG AGGGGAAGAG CCUGUACGUC 1440

AAGGGCGAGC CCAUCAUCAA CUUCUACGAC CCCCUCGUGU UCCCGUCCGA CGAGUUCGAC 1500

GCCAGCAUCU CCCAGGUGAA CGAGAAGAUC AACCAGAGCC UGGCCUUCAU CCGGAAGUCC 1560

GACGAGCUGC UGCACCACGU CAACGCCGGG AAGAGCACGA CCAACAUCAU GAUCACCACC 1620

AUCAUCAUCG UGAUCAUCGU GAUCCUCCUG UCCCUGAUCG CGGUCGGCCU CCUGCUGUAC 1680

UGCAAGGCCC GCAGCACGCC CGUGACCCUC UCCAAGGACC AGCUGAGCGG GAUCAACAAC 1740

AUCGCCUUCU CCAACUGAGG ACUAGUGCAU CACAUUUAAA AGCAUCUCAG CCUACCAUGA 1800

GAAUAAGAGA AAGAAAAUGA AGAUCAAUAG CUUAUUCAUC UCUUUUUCUU UUUCGUUGGU 1860

GUAAAGCCAA CACCCUGUCU AAAAAACAUA AAUUUCUUUA AUCAUUUUGC CUCUUUUCUC 1920

UGUGCUUCAA UUAAUAAAAA AUGGAAAGAA CCUAGAUCUA AAAAAAAAAA AAAAAAAAAA 1980

AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAUGCAUCC CCCCCCCCCC 2040

CCCCCCCCCC CCCCCCCCCA AAGGCUCUUU UCAGAGCCAC CAGAAUU 2087 (SEQ ID NO: 26)

EPO

For the preparation of the DNAi expression cassette of the present invention, the following vector encoding murine EPO was prepared:

In the pCI-neo vector, the T7 promoter followed by the 5'TOP-UTR sequence of HSD17B4, the GC-rich sequence encoding EPO (EPO(GC)), the 3'UTR of the mutated alpha globin (muag), A64 poly(A) Sequence, poly(C) sequence (C30) and histone stem-loop sequence (histone-SL) were prepared including:

HSD17B4 - EPO (GC) - muag - A64 - C30 - Histone SL

GGGAGACCAC AACGGUUUCC CUCUAGAAAG AACCA UGGGCG UGCCCGAGCG GCCGACCCUG 60

CUCCUGCUGC UCAGCCUGCU GCUCAUCCCC CUGGGGCUGC CCGUCCUCUG CGCCCCCCCG 120

CGCCUGAUCU GCGACUCCCG GGUGCUGGAG CGCUACAUCC UCGAGGCCAA GGAGGCGGAG 180

AACGUGACCA UGGGCUGCGC CGAGGGGCCC CGGCUGAGCG AGAACAUCAC GGUCCCCGAC 240

ACCAAGGUGA ACUUCUACGC CUGGAAGCGC AUGGAGGUGG AGGAGCAGGC CAUCGAGGUC 300

UGGCAGGGCC UGUCCCUCCU GAGCGAGGCC AUCCUGCAGG CGCAGGCCCU CCUGGCCAAC 360

UCCAGCCAGC CCCCGGAGAC ACUGCAGCUC CACAUCGACA AGGCCAUCUC CGGGCUGCGG 420

AGCCUGACCU CCCUCCUGCG CGUGCUGGGC GCGCAGAAGG AGCUCAUGAG CCCGCCCGAC 480

ACGACCCCCC CGGCCCCGCU GCGGACCCUG ACCGUGGACA CGUUCUGCAA GCUCUUCCGC 540

GUCUACGCCA ACUUCCUGCG GGGCAAGCUG AAGCUCUACA CCGGGGAGGU GUGCCGCCGG 600

GGCGACCGCU GACCACUAGU GCAUCACAUU UAAAAGCAUC UCAGCCUACC AUGAGAAUAA 660

GAGAAAGAAA AUGAAGAUCA AUAGCUUAUU CAUCUCUUUU UCUUUUUCGU UGGUGUAAAG 720

CCAACACCCU GUCUAAAAAA CAAUAAAUUUC UUUAAUCAUU UUGCCUCUUU UCUCUGUGCU 780

UCAAUUAAUA AAAAAUGGAA AGAACCUAGA UCUAAAAAAA AAAAAAAAAA AAAAAAAAAA 840

AAAAAAAAAA AAAAAAAAAAAAAAAAAAAA AAAAAAAUGC AUCCCCCCCC CCCCCCCCCC 900

CCCCCCCCCC CCCAAAGGCU CUUUUCAGAG CCACCAGAAU U 941 (SEQ ID NO: 27)

For comparison, a vector was prepared comprising the T7 promoter followed by the wild-type sequence encoding EPO (EPO(wt)) and the A30 poly(A) sequence:

EPO (wt) - A30

GGGAGACCAC AACGGUUUCC CUCUAGAAAG AACC ACCAUGGG GGUGCCCGAA CGUCCCACCC 60

UGCUGCUUUU ACUCUCCUUG CUACUGAUUC CUCUGGGCCU CCCAGUCCUC UGUGCUCCCC 120

CACGCCUCAU CUGCGACAGU CGAGUUCUGG AGAGGUACAU CUUAGAGGCC AAGGAGGCAG 180

AAAAUGUCAC GAUGGGUUGU GCAGAAGGUC CCAGACUGAG UGAAAAUAUU ACAGUCCCAG 240

AUACCAAAGU CAACUUCUAU GCUUGGAAAA GAAUGGAGGU GGAAGAACAG GCCAUAGAAG 300

UUUGGCAAGG CCUGUCCCUG CUCUCAGAAG CCAUCCUGCA GGCCCAGGCC CUGCUAGCCA 360

AUUCCUCCCA GCCACCAGAG ACCCUUCAGC UUCAUAUAGA CAAAGCCAUC AGUGGUCUAC 420

GUAGCCUCAC UUCACUGCUU CGGGUACUGG GAGCUCAGAA GGAAUUGAUG UCGCCUCCAG 480

AUACCACCCC ACCUGCUCCA CUCCGAACAC UCACAGUGGA UACUUUCUGC AAGCUCUUCC 540

GGGUCUACGC CAACUCUCUC CGGGGGAAAC UGAAGCUGUA CACGGGAGAG GUCUGCAGGA 600

GAGGGGACAG GUGACCACUA GUAGAUCUAA AAAAAAAAAA AAAAAAAAAA AAAAAAAA 658 (SEQ ID NO: 28)

Cancer antigen of the oncogene KRAS mutant

In vaccine development, the need for the development of cancer antigens that elicit a desired immune response (eg, T-cell response) against a targeted polypeptide sequence is very high. Using the self-transcribed RNA/DNA system of the present invention, it is possible to tailor the desired immune response by selecting the appropriate T or B cell cancer epitope and formulating the epitope or antigen for effective delivery in vivo.

The immune response can be further increased by selecting one or more universal type II T-cell epitopes to be further delivered to an appropriate T and/or B cell cancer epitope or antigen.

The RNA/DNA system of the present invention may include an activating oncogene mutant peptide (eg, a KRAS mutant peptide). Oncogenetic mutations may have the ability to elevate specific T cells. This occurs in association with the most common Human Leucocyte Antigen (HLA) allele (A2, occurring in approximately 50% of Caucasians). It is also possible to generate specific T cells for point mutations with respect to the less common HLA alleles (A11, C8). These findings have led to significant advances in cancer treatment.

Oncogene mutations are common in many cancers. The ability to target these mutations and generate sufficient T cells to kill tumors has broad applicability for cancer therapy. Delivery of antigens using the DNAi expression cassette generated in the RNA/DNA system of the present invention has these significant advantages over delivery of peptide vaccines. The present invention shows that activating oncogenic mutations delivered in vivo in the form of a DNAi expression cassette significantly improves the effectiveness of cancer therapy.

Cancer vaccines and vaccination methods using the RNA/DNA system of the present invention include epitopes or antigens (neoepitopes) based on specific mutations and those expressed by cancer-germline genes (antigens common to tumors found in multiple patients). include

An epitope, known as an antigenic determinant, is a portion of an antigen recognized by the immune system by an antibody, B cell or T cell. Epitopes include B cell epitopes and T cell epitopes. B-cell epitopes are peptide sequences required for recognition by specific antibodies to produce B-cells. A B cell epitope refers to a specific region of an antigen recognized by an antibody. The portion of an antibody that binds to an epitope is called a paratope. An epitope may be a conformational epitope or a linear epitope based on structure and interactions with the paratope. Linear or continuous, an epitope is defined by the primary amino acid sequence of a specific region of a protein.

Sequences that interact with the antibody are placed next to each other sequentially on the protein, and the epitope can usually be mimicked by a single peptide. A conformational epitope is an epitope determined by the conformational structure of a native protein. These epitopes may be contiguous or discontinuous, ie, components of the epitope may be located on heterogeneous portions of the protein that are brought into close contact with each other in the folded native protein structure.

HLA class I molecules are highly polymorphic transmembrane glycoproteins composed of two polypeptide chains (heavy chain and light chain). HLA, ie, the major histocompatibility complex in humans, is specific to each individual and has genetic characteristics. Class I heavy chains are encoded by three genes: HLA-A, HLA-B and HLA-C.

HLA class I molecules are important for establishing an immune response by presenting endogenous antigens to T lymphocytes, which initiate a chain of immune responses that result in tumor cell clearance of cytotoxic T cells.

Altered levels of HLA class I antigen production are a prevalent phenomenon in malignancies and are accompanied by significant inhibition of anti-tumor T cell function. This represents one major mechanism used by cancer cells to evade immune surveillance.

Down-regulated levels of HLA class I antigens were detected in 90% of NSCLC tumors (n=65). Reduction or loss of HLA was detected in 76% of pancreatic tumor samples (n=19). Expression of HLA class I antigens in colon cancer was dramatically reduced or undetectable in 96% of tumor samples (n=25).

Two general strategies can be applied by tumor cells to escape immune surveillance: immunoselection (poorly immunogenic variants of tumor cells) and immunosubversion (disruption of the immune system). A correlation between changes in HLA class I antigens and the presence of KRAS codon 12 mutations has been demonstrated, with a possible inducible effect of KRAS codon 12 mutations on HLA class I antigens in cancer progression. A number of frequent cancer mutations are predicted to bind to HLA class I alleles with high affinity (IC50 <= 50 nM)7 and may be suitable for prophylactic cancer vaccination.

KRAS is the most frequently mutated oncogene in human cancers (approximately 15%). KRAS mutations are best conserved in a single "hotspan" and activate oncogenes. KRAS oncogenic mutations increase specific T cells. However, most of these are associated with the most common HLA allele (A2, occurring in approximately 50% of Caucasians). More recently, (a) point mutation specific T cells can be generated with respect to the less common HLA alleles (A11, C8). and (b) mediating a dramatic tumor response in patients with lung cancer in a clinic where culturing these cells ex vivo and treating them back to the patient. (N Engl J Med 2016; 375:2255-2262 December 8, 2016 DOI: 10.1056/NEJMoa1609279).

As shown in Table 2 below, in CRC (colorectal cancer), only three mutations (G12V, G12D and G13D) account for 96% of cases. Furthermore, all CRC patients were typed for KRAS mutations as a standard of care.

The Catalog Of Somatic Mutations In Cancer (COSMIC) Case Count type all cancers % CRC % G12S 1849 One% G12V 9213 4% 5215 29% G12C 4535 2% G12D 13634 7% 8083 44% G12A 2179 One% G12R 1244 One% G13D 5084 2% 4267 23% 18% 96% gun 208629 18271 http://cancer.sanger.ac.uk/cosmic/gene/analysis?In=KRAS

In another COSMIC data set, 73.68% of KRAS mutations in colorectal cancer are accounted for by these three mutations (G12V, G12D and G13D) (Table 3).

Frequency of KRAS mutations in colorectal cancer type colon % rectal % gun % G12D 635 35.04 178 33.46 813 34.68 G12V 364 20.09 124 23.31 488 20.82 G13D 338 18.65 88 16.54 426 18.17 73.68

Exemplary KRAS mutant peptide sequences and mRNA constructs are shown in Tables 4-6.

KRAS mutant peptide sequence type 9 amino acid sequence 15-mer 25-mer G12D VVGADGVGK
(SEQ ID NO: 29) KLVVVGADGVGKSal
(SEQ ID NO: 30) MTEYKLVVVGADGVGKSalTIQLIQ
(SEQ ID NO: 31) G12V VVGAVGVGK
(SEQ ID NO: 32) KLVVVGAVGVGKSal
(SEQ ID NO: 33) MTEYKLVVVGAVGVGKSalTIQLIQ
(SEQ ID NO: 34) G13D VVGAGDVGK
(SEQ ID NO: 35) KLVVVGAGDVGKSal
(SEQ ID NO: 36) MTEYKLVVVGAGDVGKSalTIQLIQ
(SEQ ID NO: 37) G12C VVGACGVGK
(SEQ ID NO: 38) KLVVVGACGVGKSal
(SEQ ID NO: 39) MTEYKLVVVGACGVGKSalTIQLIQ
(SEQ ID NO: 40) WT MTEYKLVVVGAGGVGKSalTIQLIQ
(SEQ ID NO: 41)

KRAS mutant amino acid sequence KRAS mutant amino acid sequence KRAS(G12D)
15-mer MKLVVVGADGVGKSal (SEQ ID NO: 42) KRAS(G12V)
15-mer MKLVVVGAVGVGKSal (SEQ ID NO: 43) KRAS(G13D)
15-mer MKLVVVGADGVGKSal (SEQ ID NO: 44) KRAS(G12D)
25-mer MTEYKLVVVGADGVGKSalTIQLIQ (SEQ ID NO: 45) KRAS(G12V)
25-mer MTEYKLVVVGAVGVGKSalTIQLIQ (SEQ ID NO: 46) KRAS(G13D)
25-mer MTEYKLVVVGADGVGKSalTIQLIQ (SEQ ID NO: 47) KRAS(G12D)
15-mer ^3 MKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSal (SEQ ID NO: 48) KRAS(G12V)
15-mer ^3 MKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSal (SEQ ID NO: 49) KRAS(G13D)
15-mer ^3 MKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSal (SEQ ID NO: 50) KRAS(G12D)
25-mer ^3 MKLVVVGADGVGKSalKLVVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADG (SEQ ID NO: 51) KRAS(G12V)
25-mer ^3 MKLVVVGAVGVGKSalKLVVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVG (SEQ ID NO: 52) KRAS(G13D)
25-mer ^3 MKLVVVGADGVGKSalKLVVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADG (SEQ ID NO: 53) KRAS(G12C)
25-mer MTEYKLVVVGACGVGKSalTIQLIQ (SEQ ID NO: 54) KRAS(G12C)
25-mer ^3 MTEYKLVVVGACGVGKSalTIQLIQTEYKLVVVGACGVGKSalTIQLIQTEYKLVVVGACGVGKSalTIQLIQ (SEQ ID NO: 55) KRAS(WT)
25-mer TEYKLVVVGAGGVGKSalTIQLIQ (SEQ ID NO: 56)

KRAS mutant antigen mRNA sequence mRNA name orf sequence (amino acid) orf sequence (nucleotides) KRAS(G12D)
25-mer MTEYKLVVVGADGVGKSalTIQLIQ (SEQ ID NO: 57) ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCGACGGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAG (SEQ ID NO: 58) KRAS(G12V)
25-mer MTEYKLVVVGAVGVGKSalTIQLIQ (SEQ ID NO: 59) ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCGTGGGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAG (SEQ ID NO: 60) KRAS(G13D)
25-mer MTEYKLVVVGADGVGKSalTIQLIQ (SEQ ID NO: 61) ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCGGCGACGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAG (SEQ ID NO: 62) KRAS(G12D)
25-mer ^3 MKLVVVGADGVGKSalKLVVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADG (SEQ ID NO: 63) ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCGACGGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAGACCGAGTACAAGCTGGTGGTGGTGGGCGCCGACGGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAGACCGAGTACAAGCTGGTGGTGGTGGGCTCGCCGACGGCGTGGGCAGGATCGACGGCGTGGGCGATC (SEQ ID NO:64) KRAS(G12V)
25-mer ^3 MKLVVVGAVGVGKSalKLVVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVGVGKSalKLVVVGAVG (SEQ ID NO: 65) ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCGTGGGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAGACCGAGTACAAGCTGGTGGTGGTGGGCGCCGTGGGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAGACCGAGTACAAGCTGGTGGTGGTGGGCTCGCCGTGGGCGTGGGCAGGATCGTGGGCGTGGGCAGGATCGTGGGCGTGGGCAA KRAS(G13D)
25-mer ^3 MKLVVVGADGVGKSalKLVVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalKLVVVGADGVGKSalK67VVGADGVGKSalKLVVVGADG (SEQ ID NO: ) ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCGGCGACGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAGACCGAGTACAAGCTGGTGGTGGTGGGCGCCGGCGACGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAGACCGAGTACAAGCTGGTGGTGGTGGGCTCGCCGGCGACGTGGGCAGGATCGGCGACGTGGGCAGSEQ ID NO: 68 KRAS(G12C)
25-mer MTEYKLVVVGACGVGKSalTIQLIQ (SEQ ID NO: 69) ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCTGCGGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAG (SEQ ID NO: 70) KRAS(G12C)
25-mer ^3 MTEYKLVVVGACGVGKSalTIQLIQTEYKLVVVGACGVGKSalTIQLIQTEYKLVVVGACGVGKSalTIQLIQ (SEQ ID NO:71) ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCTGCGGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAGACCGAGTACAAGCTGGTGGTGGTGGGCGCCTGCGGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAGACCGAGTACAAGCTGGTGGTGGTGGGCTCGCCTGCGGCGTGGGCAGGAGCTGCGGCGTGGGCAGSequence #72 KRAS(WT)
25-mer TEYKLVVVGAGGVGKSalTIQLIQ (SEQ ID NO: 73) ATGACCGAGTACAAGCTGGTGGTGGTGGGCGCCGGCGGCGTGGGCAAGAGCGCCCTGACCATCCAGCTGATCCAG (SEQ ID NO: 74)

For the preparation of a DNAi expression cassette containing the cancer antigen (KRASi) of the oncogene KRAS mutant of the present invention, the following vector encoding the cancer antigen (KRASi) of multiple oncogene KRAS mutants was prepared.

As in vivo cleavable linkers, P2A was selected from the 2A self-cleaving peptides in <Table 7> and used.

Sequence of 2A self-cleaving peptides designation amino acid sequence T2A (GSG) EGRGSLL TCGDVEENPGP (SEQ ID NO: 75) P2A (GSG) ATNFSLLKQAGDVEENPGP (SEQ ID NO: 76) E2A (GSG) QCTNYalLKLAGDViNPGP (SEQ ID NO: 77) F2A (GSG) VKQTLNFDLLKLAGDViNPGP (SEQ ID NO: 78)

T2A: 5'-GGAAGCGGAG AGGGCAGAGG AAGTCTGCTA ACATGCGGTG ACGTCGAGGA GAATCCTGGA CCT-3' (SEQ ID NO: 79)

P2A: 5'-GGAAGCGGAG CTACTAACTT CAGCCTGCTG AAGCAGGCTG GAGACGTGGA GGAGAACCCT GGACCT-3' (SEQ ID NO: 80)

E2A: 5'-GGAAGCGGAC AGTGTACTAA TTATGCTCTC TTGAAATTGG CTGGAGATGT TGAGAGCAAC CCTGGACCT-3' (SEQ ID NO: 81)

F2A: 5'-GGAAGCGGAG TGAAACAGAC TTTGAATTTT GACCTTCTCA AGTTGGCGGG AGACGTGGAG TCCAACCCTG GACCT-3' (SEQ ID NO: 82)

The composition of multi_KRASi used in the present invention is *?**?*KRAS (G12D) 25-mer ^3 - P2A - KRAS (G12V) 25-mer ^3- P2A -KRAS (G13D) 25-mer ^3- P2A- KRAS (G12C) ) is a 25-mer ^3*?**?*.

multi-KRAS i:

GGGAGACCAC AACGGUUUCC CUCUAGAAAG AACC AUGACC GAGUACAAGC UGGUGGUGGU 60

GGGCGCCGAC GGCGUGGGCA AGAGCGCCCU GACCUCCAGC UGAUCCAGAC CGAGUACAAG 120

CUGGUGGUGG UGGGCGCCGA CGGCGUGGGC AAGAGCGCCC UGACCAUCCA GCUGAUCCAG 180

ACCGAGUACA AGCUGGUGGU GGUGGGCGCC GACGGCGUGG GCAAGAGCGC CCUGACCAUC 240

CAGCUGAUCC AGGGAAGCGG AGCUACUAAC UUCAGCCUGC UGAAGCAGGC UGGAGACGUG 300

GAGGAGAACC CUGGACCUAU GACCGAGUAC AAGCUGGUGG UGGUGGGCGC CGUGGGCGUG 360

GGCAAGAGCG CCCUGACCAU CCAGCUGAUC CAGACCGAGU ACAAGCUGGU GGUGGUGGGC 420

GCCGUGGGCG UGGGCAAGAG CGCCCUGACC AUCCAGCUGA UCCAGACCGA GUACAAGCUG 480

GUGGUGGUGG GCGCCGUGGG CGUGGGCAAG AGCGCCCUGA CCAUCCAGCU GAUCCAGGGA 540

AGCGGAGCUA CUAACUUCAG CCUGCUGAAG CAGGCUGGAG ACGUGGAGGA GAACCCUGGA 600

CCUAUGACCG AGUACAAGCU GGUGGUGGUG GGCGCCGGCG ACGUGGGCAA GAGCGCCCUG 660

ACCAUCCAGC UGAUCCAGAC CGAGUACAAG CUGGUGGUGG UGGGCGCCGG CGACGUGGGC 720

AAGAGCGCCC UGACCAUCCA GCUGAUCCAG ACCGAGUACA AGCUGGUGGU GGUGGGCGCC 780

GGCGACGUGG GCAAGAGCGC CCUGACCAUC CAG CUGAUCC AGGGAAGCGG AGCUACUAAC 840

UUCAGCCUGC UGAAGCAGGC UGGAGACGUG GAGGAGAACC CUGGACCUAU GACCGAGUAC 900

AAGCUGGUGG UGGUGGGCGC CUGCGGCGUG GGC AAGAGCG CCCUGACCAU CCAGCUGAUC 960

CAGACCGAGU ACAAGCUGGU GGUGGUGGGC GCCUGCGGCG UGGGCAAGAG CGCCCUGACC 1120

AUCCAGCUGA UCCAGACCGA GUACAAGCUG GUGGUGGUGG GCGCCUGCGG CGUGGGCAAG 1180

AGCGCCCUGA CCAUCCAGCU GAUCCAG 1207 (SEQ ID NO: 83)

The RNA for the expression of the cancer antigen (KRASi) of the oncogene KRAS mutant is called SM-KRASi, and the constructed structure is <T7 promoter - 5'UTR - KRASi - muag - A64 - C30 - histone SL> and SEQ ID NO: 83 .

In the pCI-neo vector, SM-KRASi DNA is 5'UTR after the T7 promoter, DNA encoding the cancer antigen (KRASi) of the oncogene KRAS mutant selected in <Table 6>, and 3'UTR of the mutated alpha globin ( muag), A64 poly(A) sequence, poly(C) sequence (C30) and histone stem-loop sequence (histone-SL).

1-4. Preparation of auto_DdRp or DNAi virus expression cassettes

The auto_DdRp DNA expression cassette or DNAi expression cassette of the present invention can be constructed using a plasmid or a viral vector. In this example, the auto_DdRp and DNAi virus expression cassettes were limited to the production of viral vectors, and the production of the remaining expression cassettes in the plasmid vector was described in detail in Examples 1-2 and 1-3 above. .

The auto_DdRp and DNAi expression cassette is a plasmid or viral vector, the auto_DdRp DNA fragment is (T7 promoter - 5'UTR - DdRp DNA (or chimeric NP868R-T7RNAP DNA) - muag - A64 - C30 - histone-SL) or the DNAi expression cassette is It was prepared by inserting (T7 promoter - 5'UTR - DNAi - muag - A64 - C30 - histone-SL ).

In addition to the pCI-neo vector (Promega, USA) that can construct a cell line stabilizing the auto_DdRp and DNAi expression cassettes prepared above as a mammalian expression vector, AdEasy Adenoviral Vector System (Agilent Technologii, USA) and ViraSafe™ Lentiviral Exprision System ( Cell Biolabs, USA) can be inserted to prepare auto_DdRp and DNAi expression cassettes.

The DNAen of the present invention may include NLS-SpCas9-HF1(N497A/R661A/Q695A/Q926A)-NLS, NLS-NgAgo-NLS and NLS-SpCas9H840A-linker-M_MLV_RT-NLS.

In this example, NLS-SpCas9H840A-linker-M_MLV_RT-NLS was used.

In the present invention, the target gene of the DNAge includes, but is not limited to, VEGF, PD-L1 and HEK3 genes. The type of guide nucleic acid is also determined according to the selected target endonuclease. sgRNA type if the selected target endonuclease is NLS-SpCas9-HF1(N497A/R661A/Q695A/Q926A)-NLS, phosphorylated ssDNA type if NLS-NgAgo-NLS, and <pegRNA type if NLS-SpCas9H840A-linker-M_MLV_RT-NLS +sgRNA> type.

In the present invention, the mRNA of interest encoded by DNAi is G protein of rabies virus (RAV-G), fusion protein F of RSV strain ATCC VR-26 long, EPO and cancer antigen of multiple oncogene KRAS mutants. (KRASi), but is not limited thereto.

<Adenovirus system>

In general, prokaryotes and prokaryotes use different promoters for expression cassettes. The expression cassette used in the self-transcribed RNA/DNA system of the present invention can be prepared by using the T7 promoter in common in prokaryotes and progressive organisms, particularly mammals, plants, and the like.

In this example, the auto_DdRp or DNAi DNA fragment was recombined in the pShuttle of the adenovirus system, and this was referred to as "auto_DdRp or DNAi adeno expression cassette". The transfection system using the capped SM DdRp RNA@LS and auto_DdRp or DNAi) adeno expression cassette was called the auto_DdRp or DNAi adeno expression system. In addition, the auto_DdRp DNA fragment was recombined in the adenovirus system and this was called "auto_DdRp adeno expression cassette". The transfection system using the capped SM DdRp RNA@LS and the auto_DdRp adeno expression cassette was called the auto_DdRp adeno expression system.

In this example, the pShuttle vector in the AdEasy Adenoviral Vector System (Agilent Technologii, USA) was used.

To prepare auto_DdRp or DNAi adeno expression cassette, *auto_DdRp or DNAi expression cassette is PCR-assigned to auto_DdRp DNA fragment T7 promoter - 5'UTR - DdRp DNA (or chimeric DdRp DNA) - muag - A64 - C30 - histone-SL ) or DNAi fragments (T7 promoter - 5'UTR - DNAi - muag - A64 - C30 - histone-SL) were prepared in large quantities.

First, the <T7 promoter - 5'UTR - DdRp DNA (or chimeric NP868R-T7RNAP DNA) - muag - A64 - C30 - histone-SL> region was amplified using the auto_DdRp expression cassette prepared in Example 1-2 as a template. At this time, both ends of the PCR product using a forward primer (5'- CTCGAG TAAT ACGACTCACT ATAG GGAGACCAC AACGGUUUCC CUCU -3', SEQ ID NO: 43) and a reverse primer (5'- GTCGAC GGT GGCTCTAAAA GAGCCTTTGG-3', SEQ ID NO: 51) XhoI/SalI recognition sequence was given.

A DNAi fragment encoding a type of mRNAi, G protein of rabies virus (RAV-G), fusion protein F of RSV strain ATCC VR-26 long, EPO, or cancer antigen (KRASi) of an oncogene KRAS mutant It was prepared by PCR method. By PCR using the auto_DdRp or DNAi expression cassette prepared in <Plasmid expression cassette> of Example 1-3 as a template, the auto_DdRp DNA fragment is T7 promoter - 5'UTR - DdRp DNA (or chimeric DdRp DNA) - muag - A64 - C30 - Histone-SL) or DNAi fragment (T7 promoter - 5'UTR - DNAi - muag - A64 - C30 - histone-SL) was prepared in large quantities. At this time, when PCR of the auto_(DdRp+DNAi) expression cassette, forward primer (5'- CTCGAG TAAT ACGACTCACT ATAG-3' SEQ ID NO: 76) and reverse primer (5'- GTCGAC AAAA AACCAACAAT AAGAAGAAAG A-3', SEQ ID NO: 77) ) to give XhoI/SalI recognition sequences to both ends of the PCR product.

The amplification product was treated with XhoI/SalI restriction enzymes, mixed with pShuttle vector linearized with XhoI/SalI restriction enzymes, and DNA ligase was added to perform a cloning reaction. 2-3 μl of the pShuttle vector cloning reaction solution was transduced into E. coli, plated on a selection plate, and incubated at 37° C. overnight. Pick 6-12 colonies and inoculate them into 3 ml of LB selection medium at 50 μg/ml each. Incubate overnight on a shaker at 37°C. Recombinant pShuttle containing auto_(DdRp+DNAi) DNA fragments was isolated using standard procedures or a commercial Qiaprep Miniprep kit (Qiagen). The recombinant plasmid was digested with XhoI/SalI and gel electrophoresis was performed to select an auto_(DdRp+DNAi) DNA fragment expression cassette clone (FIG. 11).

Production of the recombinant auto_DdRp or DNAi adenovirus of the present invention was performed according to the instructions of the AdEasy Adenoviral Vector System (Agilent Technologii, USA). First, the recombinant auto_DdRp or DNAi Adeno expression cassettes were linearized with Pme I. Linearized auto_DdRp or DNAi Adeno expression cassettes were co-transformed into BJ5183 bacteria with pAdEasy-1, a supercoil virus DNA plasmid. Transformants are selected for kanamycin resistance. Recombination was confirmed by a method using a restriction enzyme (FIG. 12). Restriction of recombinant Ad plasmid DNA with Pac I resulted in large fragments of ~30 kb and small fragments of 3.0 kb (if recombination occurred between the left arm) or 4.5 kb (if recombination occurred at the original replication site).

When recombination was confirmed, it was mass-produced using the XL10-Gold strain without recombination ability and digested with Pac I. Purified recombinant Ad plasmid DNA was digested with Pac I and inverted terminal repeat (ITR) was exposed. To the tube containing the DNA, 25 μl of ViraPack Transfection Kit's Solution I and 250 μl of Solution II were added and incubated at room temperature for 10 minutes. . Plates containing AD-293 cells in MBS-containing medium were prepared in an incubator. The DNA suspension was gently mixed by pipetting up and down to resuspend the DNA precipitate, then the DNA suspension was added dropwise to the plate to treat AD-293 (or HEK293) cells.

After the tissue culture plates were incubated in an incubator at 37° C. for 3 hours, the medium was removed from the plates and replaced with 4 ml of growth medium supplemented with 25 μM chloroquine (see medium and reagent preparation). After the plate is incubated for an additional 6 h in a 37 °C incubator, the growth medium containing 25 μM chloroquine is removed and replaced with 4 ml growth medium (chloroquine).

Culture plates were incubated at 37° C. for 7-10 days and growth medium was replenished as needed (based on media color). When the cells appeared to adhere well to the plate, the medium was replaced with 4 ml of fresh medium. Alternatively, when cells isolated from the growth medium were observed, the same volume of fresh medium was added to the existing medium and cultured. A primary virus stock was obtained from this cell culture solution.

The growth medium was carefully removed from the adenovirus producing AD-293 plates and the cells were washed once with PBS. 0.5 ml of PBS was added to each plate of cells to be harvested. Cells were collected by holding the plate at an angle and scraping the cells into a pool of PBS with a cell lifter. The cell suspension was transferred to a 1.7 ml screw cap microcentrifuge tube. The cell suspension was frozen/thawed 4 times by alternating the tubes between the dry ice-methanol bath and the 37°C water bath and vortexing briefly after each thawing. Cell debris was collected by microcentrifugation at 12,000 × g for 10 min at room temperature. The supernatant (primary virus stock) was transferred to a fresh screw cap microcentrifuge tube. This primary virus stock can be stored at -80°C for over 1 year.

The titer of the prepared primary virus stock was determined by the method of assay 1 (Korea Food and Drug Administration) of adenovirus-derived vector gene therapy, and was maintained at 10 ⁷ -0 ⁸ pfu/ml.

<Retivirus system>

In this Example, the auto_DdRp or DNAi DNA fragment obtained by PCR using the auto_DdRp or DNAi expression cassette prepared in the <Plasmid expression cassette> of Examples 1-4 in the shuttle vector of the retivirus system in this Example was recombined and this was "auto_DdRp or DNAi lenti" expression cassette". A transfection system using capped SM DdRp RNA@LS and an auto_DdRp or DNAi lenti expression cassette was referred to as an auto_DdRp or DNAi lenti expression system. In addition, the auto_DdRp DNA fragment was recombined in the retivirus system and this was called "auto_DdRp lenti expression cassette". The transfection system using the capped SM DdRp RNA@LS and the auto_DdRp lenti expression cassette was called the auto_DdRp lenti expression system.

In this example, the pSMPUW-IRES-Blasticidin Lentiviral Exprision Vector, the shuttle vector pSMPUW, from the ViraSafe ^™ Lentiviral Exprision System (Cell Biolabs, USA) was used.

To prepare the letti expression cassette, the auto_DdRp expression cassette and the mRNAi type, G protein of rabies virus (RAV-G), RSV strain ATCC VR-26 long fusion protein F, EPO and oncogene KRAS mutation A DNAi fragment encoding the body cancer antigen (KRASi) was prepared by PCR.

First, by PCR using the auto_DdRp or DNAi expression cassette prepared in the <plasmid expression cassette> of Example 1-4 as a template, the auto_DdRp DNA fragment is (T7 promoter - 5'UTR - DdRp DNA (or chimeric DdRp DNA) - muag - A64 - C30 - histone-SL) or DNAi fragment (T7 promoter - 5'UTR - DNAi - muag - A64 - C30 - histone-SL) was prepared in large quantities. At this time, BamHI / at both ends of the PCR product using a forward primer (5'- GGATCC TAAT ACGACTCACT ATAG-3' SEQ ID NO: 78) and a reverse primer (5'- GTCGAC AAAA AACCAACAAT AAGAAGAAAG A-3', SEQ ID NO: 79) SalI recognition sequence was given. A primer for ribozyme DNAi is prepared separately.

The amplification product was treated with BamHI/SalI restriction enzymes, a shuttle vector pSMPUW cloning vector linearized with BamHI/SalI restriction enzymes was mixed, and DNA ligase was added to perform a cloning reaction. 2-3 μl of the recombinant pSMPUW reaction solution was transduced into E. coli, plated on a selection plate, and incubated overnight at 37°C. Pick 6-12 colonies and inoculate each in 3 ml of LB selective medium. Incubate overnight on a shaker at 37°C. Recombinant pSMPUW shuttle vectors containing auto_(DdRp+DNAi) DNA fragments were isolated using standard procedures or a commercial Qiaprep Miniprep kit (Qiagen). The recombinant vector was digested with BamHI/SalI and gel electrophoresis was performed to select an auto_(DdRp+DNAi) DNA lenti expression cassette clone (FIG. 13).

The recombinant auto_DdRp or DNAi Lenti expression cassette virus production of the present invention was performed according to the instructions of the pSMPUW Universal Lentiviral Exprision Vector (Cell Biolabs, USA) and Lipofectamine LTX Reagent (ThermoFisher, USA) as follows.

One day before transfection, plate enough 293T cells or 293LTV cells (Cat. # LTV-100) and incubate to cover 70-80% of the culture vessel on the day of transfection. The molar ratio of pSMPUW, envelope and packaging plasmids prepared in the present invention as a transfer plasmid to Lipofectamine ^™ Plus (Invitrogen), that is, pSMPUW: pCMV-VSV-G: pRSV-REV: pCgpV vector Cells are transfected by mixing 3:1:1:1.

Harvest the lentiviral supernatant 36-72 hours after transfection. The supernatant can be harvested 2-3 times every 12 hours. Store at 4°C during the collection period. Collect the collected supernatant and centrifuge at 1500 rpm for 5 min to remove cell debris and filter at 0.22 µm. The supernatant can be used directly or purified/concentrated if necessary. For long-term storage, aliquot the supernatant and store at -80°C.

The harvested and stored lentivirus solution was highly concentrated in virus using the ViraBind ^™ Lentivirus Concentration and Purification Kit (Cat. No. VPK-090, Cell Biolabs, USA) before transfection into host cells. To ensure consistent viral transduction into host cells for the auto_(DdRp+DNAi) lentivirus of the present invention, lentiviruses using p24 ELISA, QuickTiter ^™ Lentivirus Titer Kit (Cat. No. VPK-107, Cell Biolabs, USA) The titer was measured and the titer of the purified and concentrated virus stock was maintained at 10 ⁶ -0 ⁷ pfu/ml.

It is difficult for the target cells to be infected with the auto_(DdRp+DNAi) lentivirus of the present invention, and the transduction efficiency may be low without an auxiliary reagent. ViraDuctin ^™ Lentivirus Transduction Kit (catalog number LTV-200, Cell Biolabs, USA) can be used to promote virus and cell interaction to increase transduction efficiency.

<Binary vector for plant transfection>

In this example, the auto_(DdRp+DNAi) DNA fragment was recombined in a binary vector (Ti plasmid) for plant transfection, and this was called “auto_(DdRp+DNAi) Ti expression cassette”. The transfection system using the capped DdRp RNA@LS and auto_(DdRp+DNAi) Ti expression cassette was called the auto_(DdRp+DNAi) Ti expression system.

T7 promoter encoding the auto_DdRp expression cassette and the rabies virus G protein (RAV-G), the fusion protein F of RSV strain ATCC VR-26 long, EPO and the cancer antigen (KRASi) of the oncogene KRAS mutant To prepare an auto_(DdRp+DNAi) Ti expression cassette containing a DNAi fragment with

First, in this embodiment, the DNAi fragment was prepared in the <T7 promoter-5'UTR-EPO-Nos-termination sequence> structure (Biosynthiis, USA). EcoRI/XhoI sites were added to both ends by PCR. Instead of the above EPO, DNAi encoding various mRNAi including the mRNA of Examples 1-3 may be inserted.

For the preparation of the <T7 promoter-5'UTR-EPO-Nos-termination sequence> structure having EcoRI/XhoI sites at both ends, the DNA fragment synthesized earlier was amplified by PCR as a template. At this time, EcoRI / XhoI recognition sequence at both ends using a forward primer (5'- GAATTC TAAT ACGACTCACT ATAG-3', SEQ ID NO: 72), and a reverse primer (5'- CTCGAG GAT CAGTAACAT AGATG-3', SEQ ID NO: 73) A PCR product with this was obtained.

Nos- Termination sequence:

GATCGTTCAA ACATTTGGCA ATAAAGTTTC TTAAGATTGA ATCCTGTTGC CGGTCTTGCG 60

ATGATTATCA TATAATTTCT GTTGAATTAC GTTAAGCATG TAATAATTAA CATGTAATGC 120

ATGACGTTAT TTATGAGATG GGTTTTTATG ATTAGAGTCC CGCAATTATA CATTTAATAC 180

GCGATAGAAA ACAAAATATA GCGCGCAAAC TAGGATAAAT TATCGCGCGC GGTGTCATCT 240

ATGTTACTAG ATC 253

Referring to Example 1-2, an auto_DdRp DNA fragment (T7 promoter - 5'UTR - DdRp DNA - Nos termination sequence) having an XhoI/SalI site at the end was prepared and amplified by PCR. DdRp DNA sequence Alternatively, NP868R-T7RNAP DNA may be inserted. At this time, a forward primer (5'- CTCGAG TAAT ACGACTCACT ATAG-3', SEQ ID NO: 72) and a reverse primer (5'- GTCGAC GAT CAGTAACAT AGATG-3', SEQ ID NO: 73) were used at both ends of the XhoI / SalI recognition sequence A PCR product with this was obtained.

The prepared DNAi fragment and auto_DdRp DNA fragment were digested with XhoII restriction enzyme, ligated with ligase, and the auto_(DdRp+DNAi) fragment (T7 promoter - 5'UTR - DdRp DNA - Nos termination sequence with EcoRI/SalI sites at both ends). - T7 promoter - 5'UTR - EPO - Nos-termination sequence) was prepared.

This was cloned into the EcoRI/SalI site in pCAMBIA1305.1 (AbcAm, USA) and designated as the auto_(DdRp+DNAi) Ti expression cassette. In addition, a binary vector (Ti plAsmid) containing an auto_DdRp DNA fragment was referred to as an auto_DdRp Ti expression cassette.

2-3 μl of the recombinant vector cloning reaction solution was transduced into E. coli, plated on a selection plate, and incubated at 37° C. overnight.

6-12 colonies are picked and inoculated into 3 ml of 50 μg/ml LB selection medium, respectively. Incubate overnight on a shaker at 37°C. Plasmid DNA was isolated using standard procedures or a commercial Qiaprep Miniprep kit (Qiagen). Recombinant binary vector (Ti plAsmid) was digested with EcoRI/SalI and gel electrophoresis was performed to screen auto_(DdRp+DNAi) Ti expression cassette clones (FIG. 14).

The auto_(DdRp+DNAi) Ti expression cassette thus obtained was transferred to E. coli DH5α and amplified. The auto_(DdRp+DNAi) Ti expression cassette was then introduced into AGrobActerium tumefAciens LBA4404 using a freeze-thaw method to prepare an auto_(DdRp+DNAi) Ti expression system.

The auto_(DdRp+DNAi) Ti expression cassette of the present invention can be transformed into an autologous lineage of tomato plants through the A. tumefAciens LBA4404 mediated method.

1-5. Preparation of vector for nuclear-dependent mRNA expression based on CMV promoter

pCI_DNAi capable of expressing mRNAi by CMV immediAte-early enhAncer/promoter reGion and β-Globin/IGG chimeric intron in the nucleus was prepared using pCI-neo Mammalian Exprision Vector (Promega, USA).

First, the DNAi fragment encoding the mRNAi containing the XhoI recognition sequence (CTGGAC) at the 5' end and the XbaI recognition sequence (TCTAGA) at the 3' end was prepared by PCR using the DNAi fragment prepared in Example 1-3 as a template. prepared.

The DNAi prepared in this Example is DNA encoding the cancer antigens of RAV-G, RSV-F, EPO, and the oncogene KRAS mutant. It is a configuration with a late SV40 polyadenylation signal downstream of the DNAi encoding the mRNAi used.

This dsDNA was treated with XhoI/XbaI restriction enzyme and then inserted into the XhoI/XbaI site of the multicloning site of pCI-neo plasmid (Origene, USA) using T4 DNA ligase (FIG. 15).

Example 2. Preparation of liposome delivery system

A liposome delivery system was prepared by using the linear DdRp RNA expression cassette prepared in the present invention, various types of circular plasmid expression cassettes, and combinations thereof with Lipofectamine 2000 Transfection Reagent (ThermoFisher, USA).

1 μg of the mixture of the prepared DdRp RNA expression cassette, plasmid expression cassette, and a combination thereof (same molar ratio) and 100 μl of Opti-MEM were mixed in a 1.5 ml tube, and 5 μl of Lipofectamin2000 and 100 μl of serum-free medium were mixed with another 1.5 ml After mixing in a tube, it was left at room temperature for 5 minutes. Thereafter, the contents of the two tubes were mixed and stored at room temperature for 20 minutes to form a liposome-type complex. After 20 minutes, the tube was centrifuged for 10 seconds, treated and transfected on each cell, and replaced with a new medium after 4 hours.

*?**?*auto(-)_DNAi@LS*?**?*, which is a complex of the DdRp RNA/DNAi system and liposome, "DdRp RNA/DNAi@ LS". T7 polymerase repeatedly synthesized from the stabilized capped CM_DdRp mRNA can be maintained at high concentrations in the cytoplasm for a long time.

In addition, the DdRp RNA/auto_DdRp+DNAi system or the DdRp RNA/auto_(DdRp+DNAi) system and auto_DNAi@LS, which is a complex of a liposome, is a DdRp RNA expression cassette, an auto_DdRp expression cassette, and “DdRp RNA, a complex of a DNAi expression cassette and liposome” /auto_DdRp+DNAi@LS or DdRp RNA expression cassette, DdRp RNA/auto_(DdRp+DNAi)@LS, a complex of auto_(DdRp+DNAi) expression cassette and liposome. By binding to the T7 promoter of the auto_DdRp expression cassette or auto_(DdRp+DNAi) expression cassette by T7 polymerase synthesized from initially stabilized capped CM DdRp mRNA, T7 polymerase can be maintained at high concentrations in the cytoplasm for a long time as a result of autotranscription.

As a control, DOPC liposome nazoparticles loaded with pCI-neo_DNAi were prepared, and were called pCI-neo_DNAi@LS, a complex of pCI-neo_DNAi and liposomes.

Example 3. Transfection of expression-repressed RNA/DNA system

The self-transcribed RNA/DNA system of the present invention is composed of an RNA component and a DNA component, and the carrier thereof can be variously used, such as a linear nucleic acid fragment, a circular plasmid, and a virus, or a combination thereof. Various methods can be selected when the carrier of the prepared self-transcribed RNA/DNA system is non-viral. In this example, the liposome type was selected, but the present invention is not limited thereto.

Transfection of the target cells of the expression-suppressing RNA/DNA system of the present invention was performed as follows in order to smoothly obtain stabilized transformed cells. Of course, transformation can be performed on the same.

The first transfection is a step in which an expression cassette containing DNAi encoding the mRNA of interest is transfected and transformed cells are selected using a selective medium, and the second transfection is the cell stabilized in the selective medium by the first transfection. In this step, a DdRp RNA expression cassette is introduced into the target to express the mRNA of interest and the protein they encode by the DdRp synthesized in the transformed cell.

<1st transfection>

- Plasmid carrier

In the case of the plasmid delivery system, according to the instructions of pCI-neo Mammalian Exprision Vector (Promega, USA) and Lipofectamine 2000 Transfection Reagent (ThermoFisher, USA), target cells were prepared and 1x10 ⁵ cells were seeded into 96-wells. The next day, the liposome gene delivery system prepared in Example 2 was prepared in a total volume of 100 μl with a cell culture medium, and then treated in each well. 37 ℃, 5% CO ₂ After culturing for 24 hours in an incubator, the cells were washed with 1X PBS, treated with trypsin to remove the cells, and then transferred to a 100 mm culture dish and incubated.

For transfection of the plasmid delivery system, transfection can be carried out by making the RNA/DNA system together as a liposome delivery system, or by dividing them, each liposome can be produced and each transfected.

For the selection of stable transformed cells by the liposome delivery system, after transfection, the transfected cells were selected with antibiotic G-418. After transfection, cells were inoculated at a low cell density, and G-418 antibiotics were added at a concentration of 400 to 600 μg/ml for selection, and at a G-418 concentration of 200 to 400 μg/ml to maintain stable transformants. processed.

- virus carrier

In the case of the viral carrier, 1x10 ⁵ target cells were prepared according to the instructions of AdEasy Adenoviral Vector System (Agilent Technologii, USA), pSMPUW Universal Lentiviral Exprision Vector (Cell Biolabs, USA) and Lipofectamine 2000 Transfection Reagent (ThermoFisher, USA). Dog cells were seeded into 96-wells. According to the multiplicity of infection (MOI) to be treated in the cells the next day, the viral gene carrier was made into a cell culture medium in a total volume of 100 μl, and then treated in each well. After treatment with the viral gene carrier, after culturing for 24 hours at 37° C., 5% CO 2 in an incubator, the cells were washed with 1X PBS, and the cells were removed by treatment with trypsin and then transferred to a 100 mm culture dish and cultured.

In the case of lentivirus, the medium containing the antibiotic blasticidin at a concentration of 5 μg/ml was changed once every 2 to 3 days.

<Secondary transfection>

The second transfection is a step of transfecting the first transfected cells with the DdRp RNA@LS prepared in Example 2 to provide the first T7 Polymerase acting on the T7 promoter. DdRp RNA@LS transfection was performed on the cells immediately after inoculation of the selected stabilized cells and adenovirus gene carrier after treatment with the plasmid and the lentiviral carrier of the first transfection.

Example 4. T7 polymerase expression persistence experiment

Controls of the present invention 5 pmol pCI-neo_RSV-F@LS, 5 pmol DdRp RNA/RSV-F DNAi@LS, 5 pmol DdRp RNA/auto_DdRp/RSV-F DNAi@LS, 5 pmol DdRp RNA/auto_(DdRp/RSV) -F DNAi)@LS, 3x10 ⁴ MOI auto_(DdRp/RSV-F DNAi) adeno expression system [system consisting of DdRp RNA@LS and auto_(DdRp/RSV-F DNAi) adeno expression cassette] or 10 ⁴ MOI auto_( DdRp/RSV-F DNAi) lenti expression system [system consisting of DdRp RNA@LS and auto_(DdRp/RSV-F DNAi) lenti expression cassette] was transfected into 293T cells by the method of Example 3, respectively, 0, 24 , T7 polymerase mRNA and protein in 293T cells were analyzed at 48 and 72 hours. In this example, auto_DdRp of DdRp was used as a self-transcribed RNA/DNA system with T7RNAP or NP868R-T7RNAP.

- T7 polymerase mRNA analysis

For T7 polymerase mRNA analysis of the transgenic 293T cells, total RNA was isolated from 293T cells at 0, 24, 48 and 72 hours after the second transfection and analyzed by quantitative PCR (qPCR) with SYBR Green.

Specifically, total RNA was extracted from the transfected 293T cells using the RNeAsy mini kit (Qiagen). A total of 150 ng of total RNA was reverse transcribed into cDNA (SuperScript II Reverse Transcriptase, Invitrogen), and the cDNA was used for analysis using KAPA SYBR FAST qPCR Kits (Sigma aldrich, USA).

Before preparing the qPCR reaction, KAPA PROBE FAST Bio-Rad iCycler ™ qPCR Master Mix (2X), template DNA, primers and probes were thoroughly mixed as indicated in Table 8.

qPCR constitutive reagents Final Concentration 20 μl rxn PCR grade water up to 20 μl 7.2 μl qPCR Master Mix (2X) 1X 10 μl Forward Primer (10 μM) 200 nM 0.4 μl Reverse Primer (10 μM) 200 nM 0.4 μl Template DNA (10 ng/1 μl) 1 ng/μl 2.0 μl

Forward primer 5'-TCACGACTCC TTCGGTACCA T-3' (SEQ ID NO: 90) and reverse primer 5'-CATAGTTTCG CGCACTGCTT T-3' (SEQ ID NO: 91) were used.

The qPCR protocol is a Bio-Rad iCycler (Bio -Rad, USA) and analyzed the data according to the manufacturer's instructions of the equipment used. In this example, the highest expression value, the value obtained at 72 hours of DdRp RNA/auto_DdRp/RSV-F DNAi@LS, was 100%, and the result of expressing the value as a percentage in proportion to it is shown in <Fig. 17>. The experiment was performed 5 times and the average value was indicated.

As in <Fig. 16>, as a result of analyzing the DdRp of auto_DdRp transformed cells using T7RNAP, there was no expression of T7 polymerase mRNA in the control pCI-neo_RSV-F@LS, and DdRp RNA/RSV-F DNAi@LS transformed cells It was confirmed that the transfected 5'Cap structure and the stabilized DdRp RNA were slowly degraded in the cell, DdRp RNA/auto_DdRp/RSV-F DNAi@LS; DdRp RNA/auto_(DdRp/RSV-F DNAi)@LS; In cells transformed with the auto(+) DdRp expression system including the auto_(DdRp/RSV-F DNAi) adeno expression system or the auto_(DdRp/RSV-F DNAi) lenti expression system, the first It was confirmed that the T7 polymerase binds to the T7 promoter of the auto_DdRp expression cassette, and the self-transcription loop that synthesizes DdRp in the downstream region acts to stably synthesize DdRp.

When auto_DdRp of DdRp was compared with transformant cells having T7RNAP or NP868R-T7RNAP, NP868R-T7RNAP transformed cells had a 5-6 times higher expression level of T7 polymerase mRNA than T7RNAP on average, which is thought to be the effect of NP868R. became (data not shown).

- T7 polymerase assay

The transfected 293T cells were collected at 0, 24, 48 and 72 hours, lysed with RIPA buffer containing cocktail protease inhibitor, and then protein samples were prepared by centrifugation. After coating these samples on an ELISA plate at 4°C for 12 hours and blocking in 0.2% Tween-20 and 5% dry milk to prevent non-specific staining, Anti-T7 RNA Polymerase antibody (CABT-B8990, Creative diagnostic, UK), secondary staining with HRP-conjugated secondary antibody, and analysis with Promega™ Microplate Reader (Promega, USA). The experiment was performed 5 times and the average value was indicated.

As in <Fig. 17>, as a result of analyzing the DdRp of auto_DdRp transformed cells using T7RNAP, in conclusion, DdRp RNA/RSV-F DNAi@LS auto(-) DdRp expression system and DdRp RNA/auto_DdRp/RSV-F DNAi@LS; DdRp RNA/auto_(DdRp/RSV-F DNAi)@LS; T7 polymerase synthesized in cells transformed with an auto_(DdRp/RSV-F DNAi) adeno expression system or an auto(+) DdRp expression system including an auto_(DdRp/RSV-F DNAi) lenti expression system is at least in the cytoplasm. It is maintained at a high concentration for more than 72 hours.

As a result of comparing the auto_DdRp DdRp transformant cells with T7RNAP or NP868R-T7RNAP, NP868R-T7RNAP transformed cells had a higher expression level of T7 polymerase on average about 10 to 15 times than that of T7RNAP, which was thought to be the effect of NP868R. (data not shown).

Example 5. Self-transcribed RNA/DNA system comprising RSV-F mRNA

Inventive control 5 pmol pCI-neo_RSV-F @LS; 5 pmol DdRp RNA/RSV-F DNAi@LS; 5 pmol DdRp RNA/auto_DdRp/RSV-F DNAi@LS; 5 pmol DdRp RNA/auto_(DdRp/RSV-F DNAi)@LS; 3x10 ⁴ MOI auto_(DdRp/RSV-F DNAi) adeno expression system [system consisting of DdRp RNA@LS and auto_(DdRp/RSV-F DNAi) adeno expression cassette]; or 10 ⁴ MOI auto_(DdRp/RSV-F DNAi) lenti expression system [system consisting of DdRp RNA@LS and auto_(DdRp/RSV-F DNAi) lenti expression cassette]; as described in Example 3, each 293T The levels of target RSV-F mRNA and protein expression were analyzed at 0, 24, 48 and 72 hours after transfection into cells.

In this example, DdRp of auto_DdRp is a result of comparing transformed cells using NP868R-T7RNAP.

- Investigation of transformation of RSV-F mRNA expression in cells

The DdRp RNA/RSV-F DNA@LS of the present invention, auto(-)_mRNAi@LS, is the first T7 polymerase repeatedly synthesized from externally introduced stabilized and capped CM/SM DdRp mRNA, maintained at a high concentration in the cytoplasm for a long period of time. By repeatedly binding to the T7 promoter of the RSV-F DNAi expression cassette, RSV-F mRNAi expression can be maintained continuously for a long period of time.

DdRp RNA/auto_DdRp/RSV-F DNAi@LS of the present invention; DdRp RNA/auto_(DdRp/RSV-F DNAi)@LS; auto_(DdRp/RSV-F DNAi) adeno expression system; or (DdRp/RSV-F DNAi) lenti expression system; by binding to the T7 promoter of the auto_DdRp expression cassette or the auto_DdRp+DdRp RNA expression cassette by the first T7 polymerase synthesized from the initially stabilized capped CM/SM_DdRp mRNA. Polymerase is maintained at a high concentration in the cytoplasm for a long period of time as a result of self-transcription, and the T7 polymerase repeatedly binds to the T7 promoter of the DNAi expression cassette so that RSV-F mRNAi expression can be maintained continuously for a long time.

Total RNA was isolated from the transformants of the experimental group and control transformants of the present invention at 0, 24, 48 and 72 hours and reverse transcribed into cDNA (SuperScript II Reverse Transcriptase, Invitrogen), and KAPA SYBR FAST for the cDNA Analysis was performed using qPCR Kits (Sigma aldrich, USA).

Before preparing the qPCR reaction, RSV-F mRNA was investigated by qPCR by thoroughly mixing KAPA PROBE FAST Bio-Rad iCycler ™ qPCR Master Mix (2X), template DNA, primers and probes as presented in Table 3.

Specifically, total RNA was extracted from transfected 293T cells using the RNeAsy mini kit (Qiagen). A total of 150 ng of total RNA was reverse transcribed into cDNA using random hexameric primers and multiple scribe reverse transcriptase at 48° C. for 30 min.

Forward primer 5'-GCGTGATGAACTTCGAGGA-3' (SEQ ID NO: 86) and reverse primer 5'-CAATAGTGATGACCTGGCCGT-3' (SEQ ID NO: 87), which can be matched to RSV-F mRNAi, were used.

The qPCR protocol is a Bio-Rad iCycler (Bio -Rad, USA) and analyzed the data according to the manufacturer's instructions of the equipment used. In this example, the highest expression value, the value obtained at 72 hours of DdRp RNA/auto_DdRp/RSV-F DNAi@LS, was 100%, and the result of expressing the value as a percentage in proportion to it is shown in <FIG. 18>. The experiment was performed 5 times and the average value was indicated.

As in <Figure 18>, compared to the control pCI-neo_RSV-F @LS DdRp RNA / RSV-F DNAi@LS of the present invention; DdRp RNA/auto_DdRp/RSV-F DNAi@LS; DdRp RNA/auto_(DdRp/RSV-F DNAi)@LS; auto_(DdRp/RSV-F DNAi) adeno expression system; or auto_(DdRp/RSV-F DNAi) lenti expression system; cells treated with RSV-F mRNAi were observed to increase for 24 hours, and high concentrations of RSV-F mRNAi could be observed for at least 9 days at 48 hours, In the cells treated with the control pCI-neo_RSV-F @LS, the mRNAi concentration was similar to that of the cells treated with auto_mRNAi@LS, the experimental group, for 24 hours. Measurements were possible up to 72 hours.

These results show that the expression of RSV-F mRNA in the cytoplasm using the transformed cells of the experimental group of the present invention is achieved over a long period of time, and RSV-F mRNA is effectively It shows that the molecules are transformed.

- Investigation of RSV-F protein changes in cells

Control of the present invention 5 pmol pCI-neo_RSV-F DNAi @LS, Experimental group 5 pmol DdRp RNA/RSV-F DNAi@LS, 5 pmol DdRp RNA/auto_DdRp/RSV-F DNAi@LS, 5 pmol DdRp RNA/auto_(DdRp /RSV-F DNAi)@LS, 3x10 ⁴ MOI auto_(DdRp/RSV-F DNAi) adeno expression system [system consisting of DdRp RNA@LS and auto_(DdRp/RSV-F DNAi) adeno expression cassette] or 10 ⁴ MOI The auto_(DdRp/RSV-F DNAi) lenti expression system [a system consisting of DdRp RNA@LS and an auto_(DdRp/RSV-F DNAi) lenti expression cassette] was transfected into 293 T cells as described in Example 3, respectively. Afterwards, the levels of target mRNA and protein expression of multiple shRNAs were analyzed at 0, 24, 48 and 72 hours. In this example, auto_DdRp DdRp is a result of comparing transformed cells having NP868R-T7RNAP.

The amount of modification of the RSV-F protein of interest was measured after 0.24, 48 and 72 hours of treatment in the experimental group and control 293 T cells of the present example.

RSV-F synthesis was investigated in cells transformed with the control pCI_mRNAi@LS and the experimental group using ELISA using an anti-RSV-F antibody (ab94968, abcam, USA). 293T cells were treated and the amount of RSV-F strain was measured after 0.24.48 and 72 hours had elapsed. After treatment with the control pCI_mRNAi@LS (final concentration of 50 nM mRNAi), ELISA was performed to measure the amount of modification of RSV-F after the same time elapsed. Existing nuclear-dependent mRNA expression system (pCI_mRNAi@LS) and DdRp RNA/RSV-F DNAi@LS, DdRp RNA/auto_DdRp/RSV-F DNAi@LS and DdRpRNA/auto_(DdRp/RSV-F DNAi)@LS of the present invention It shows the results of comparing how different the duration of the effect of increasing protein synthesis is compared. Specifically, compared to control pCI_mRNAi@LS, DdRp RNA/RSV-F DNAi@LS, DdRp RNA/auto_DdRp/RSV-F DNAi@LS, DdRpRNA/auto_(DdRp/RSV-F DNAi)@LS, auto_(DdRp/RSV) -F DNAi) In the experimental group including the adeno expression system or the auto_(DdRp/RSV-F DNAi) lenti expression system, the increased amount of RSV-F in the cells was measured. The experiment was performed 5 times and the average value was indicated.

As shown in <Fig. 19>, auto(-)_mRNAi@LS, which is DdRp RNA/RSV-F DNAi@LS of the present invention, is transformed by T7 polymerase repeatedly synthesized from the stabilized capped CM/SM DdRp mRNA. The RSV-F protein was stably and continuously provided because RNA expression could be maintained for a long period of time by maintaining high concentration in the cytoplasm of 293 T cells and repeatedly binding to the T7 promoter of the DdRp RNA expression cassette.

In the experimental group of the present invention, by binding to the T7 promoter of the auto_DdRp expression cassette or the auto_DdRp + DdRp RNA expression cassette by the first T7 polymerase synthesized initially from the stabilized capped CM/SM DdRp mRNA, T7 polymerase is transformed as a result of self-transcription It is maintained at a high concentration for a long time in the cytoplasm of 293 T cells, and the T7 polymerase repeatedly binds to the T7 promoter of the DdRp RNA expression cassette, so that RSV-F mRNAi expression can be maintained continuously for a long period of time, thereby stably and continuously providing the RSV-F. did

These results show that the limitations of increasing short protein synthesis of the existing nuclear-dependent mRNA expression system are as follows: DdRp RNA/RSV-F DNAi@LS, DdRp RNA/auto_DdRp/RSV-F DNAi@LS, DdRpRNA/auto_(DdRp/RSV- It shows that the experimental group including F DNAi)@LS, auto_(DdRp/RSV-F DNAi) adeno expression system or auto_(DdRp/RSV-F DNAi) lenti expression system can solve effectively.

Example 6. Self Transcription RNA/DNA System Containing EPO

Controls of the present invention 5 pmol pCI-neo_EPO@LS, 5 pmol DdRp RNA/EPO DNAi@LS, 5 pmol DdRp RNA/auto_DdRp/EPO DNAi@LS, 5 pmol DdRp RNA/auto_(DdRp/EPO DNAi)@LS, 3x10 ⁴ MOI auto_(DdRp/EPO DNAi) adeno expression system [system consisting of DdRp RNA@LS and auto_(DdRp/EPO DNAi) adeno expression cassette] or 10 ⁴ MOI auto_(DdRp/EPO DNAi) lenti expression system [DdRp RNA@ LS and auto_(DdRp/EPO DNAi) lenti expression cassette] were transfected into 293T cells, respectively, as described in Example 3, and the level of EPO protein expression was analyzed at 0, 24, 48 and 72 hours. . In this example, auto_DdRp DdRp is a result of comparing transformed cells having NP868R-T7RNAP.

Total protein was isolated at 0, 24, 48 and 72 hours after treatment in the transformant 293T cells by the control group and the unconstitutional group, and the EPO level was determined by ELISA (mouse EPO QuAntikine ELISA kit, R&D Systems), and the The result is shown in <Fig. 20>. The experiment was performed 5 times and the average value was indicated.

Example 7. Self-transcription RNA/DNA system comprising multi_KRASi

- Investigation of protein changes in cells

multi_KRASi used in the present invention is <KRAS(G12D) 25-mer ^3 - P2A - KRAS(G12V) 25-mer ^3- P2A -KRAS(G13D) 25-mer ^3- P2A - KRAS(G12C) 25-mer ^3> was composed

The expression of DdRp RNA/multi_KRASi DNA@LS nanoparticles containing multi_KRASi of the present invention is Expression efficiency was investigated by transforming 293T cells with 1 μg of each nanoparticle. DdRp RNA/multi_KRASi DNA@LS nanoparticles comprising multi_KRASi of the present invention are experimental group DdRp RNA/multi_KRASi DNA@LS, DdRp RNA/auto_DdRp/multi_KRASi DNA@LS or DdRp RNA/auto_(DdRp/multi_KRASi DNA)@LS and control group pCI_neo multi_KRASi@LS was used. In this example, the result of comparing transformant cells having NP868R-T7RNAP with DdRp of auto_DdRp.

After 24 h, cell lysates were prepared by treatment of cells with RIPA lysis buffer (0.22M NACl, 0.38M Tris-HCl, pH 7.5, 0.25% sodium deoxycholate and 1% IGEPal-630). Protein concentration was measured using the Micro BCA Protein Assay Reagent Kit (Pierce, USA). Polyvinylidenefluoride membranes were incubated overnight at 4°C with primary antibody in Tween-Tris-buffered saline containing 1% bovine serum albumin.

The secondary antibody was then incubated with the membrane for 1 hour at room temperature, then the membrane was washed extensively for 30 minutes with Tween-Tris buffered saline at room temperature. Blots were investigated with the ECL Witern Blot Detection System (Millipore, USA) and visualized with the BioSpectrum AC ImAGing System (UVP, USA) according to the manufacturer's instructions.

Antibodies used for Western blotting were Anti-Kras (F234) (sc-30; Santa Cruz Biotechnology, USA), Anti-tubulin (CD66; OncoGene Science, USA), Anti-β-actin (clone mAb1501; Chemicon, USA). ), Anti-p44/42 MAPK (T202/Y204) (D13.14.4E; Cell Signaling Technology, USA) and Anti-44/42 MAPK (ERK1/2) (137F5; Cell Signaling Technology, USA) and the like.

Expression of multi_KRASi was confirmed in 293T cells transformed with the experimental group RNA/DNA1_multi_KRASi@LS, RNA/DNA2_multi_KRASi@LS or RNA/DNA3_multi_KRASi@LS and the control group pCI_multi_KRASi@LS.

<FIG. 21> shows <KRAS (G12D) 25 mer ^ 3 - P2A - KRAS (G12V) 25 mer ^ 3 - P2A - KRAS (G13D) 25 mer ^ 3 - P2A - KRAS (G12C) 25 mer ^ Multi_KRASi composed of 3> was expressed as a precursor and cleaved from the P2A domain as a subsequent operation in 293T cells, KRAS (G12D) 25-mer ^3, KRAS (G12V) 25-mer ^3, KRAS (G13D) 25-mer ^3, and KRAS It was confirmed that (G12C) 25-mer ^3 was formed.

<FIG. 22> shows 293T cells transformed with experimental groups RNA/DNA1_multi_KRASi@LS, RNA/DNA2_multi_KRASi@LS or RNA/DNA3_multi_KRASi@LS by Western blotting. Inactivation of RAs activity was demonstrated by loss of phospho-ERK, a downstream ERK activity. In <FIG. 22>, the inactivation of RAs activity in 293T cells transformed with the experimental group RNA/DNA3_multi_KRASi@LS and the control complex DdRp RNA@LS was confirmed as a loss of phospho-ERK, a downstream ERK activity.

- Prevention and treatment investigation

To confirm the prophylactic antitumor effect of the RNA/DNA1_multi_KRASi@LS, RNA/DNA2_multi_KRASi@LS, or RNA/DNA3_multi_KRASi@LS nanoparticles of the present invention, the nanoparticles prepared at 10-day intervals were applied to the right of 6-7 week old BalB/c mice. An immunized tumor model was prepared by subcutaneous injection in the anterior flank three times. In the prophylactic antitumor experiment of the immunized tumor model, CT26 cells (3×10 ⁵ /per mouse) or (1×10 ⁵ /per mouse) were injected into the right anterior flank of female BalB/c mice one week after the third administration. Administered via subcutaneous injection (n = 5-8 per group). The control group was prepared by administering PBS to the right anterior flank of female BalB/c mice.

To evaluate the therapeutic effect of the RNA/DNA1_multi_KRASi@LS, RNA/DNA2_multi_KRASi@LS or RNA/DNA3_multi_KRASi@LS nanoparticles of the present invention, 2×10 ⁵ CT26 cells were subcutaneously administered to 6-7 week old BalB/c mice and After two days, mice were randomly divided into 4 groups (n = 5-8). The prepared nanoparticles were administered to two groups a total of 3 times with an interval of 1 week. The remaining two groups were administered PBS and used as a control group.

Tumor development was monitored every 2 or 3 days, and two-dimensional measurements were recorded using a Vernier caliper. Tumor size was determined according to Park Jung-sik, tumor size = 0.5 x length x (width) ² : A tumor volume reaching 2,000 mm ³ was recorded as death and the mouse was euthanized. Tumor growth and survival curves were created and analyzed.

A 5-week-old female BalB/c mouse was purchased (SLC, Japan), acclimatized for 1 week, and then used in the experiment. The colorectal cancer cell line (CT26) was prepared in RPMI-1640 medium (GIBCO) supplemented with 10% (v/v) fetal bovine serum (FBS; GIBCO) and 1% streptomycin-penicillin (P/S) at 37°C in 5% CO ₂ was maintained.

<Figure 23> shows the protective effect of RNA/DNA1_multi_KRASi@LS, RNA/DNA2_multi_KRASi@LS and RNA/DNA3_multi_KRASi@LS nanoparticles on tumorigenesis after administration of two different doses of CT26 cells in an immune tumor model. The flow chart of immunization and tumor inoculation is as shown in <Fig. 23a>, and immunized mice were prepared by administering nanoparticles three times at 10-day intervals. Two different doses of CT26 cells were subcutaneously injected into the immunized mice, and 3x10 ⁵ CT26 cells/mouse was administered to the right flank of female BalB/c mice (n = 5-8) one week after the third nanoparticle administration. . The tumor volume was determined in individual mice and the results calculated for each group were as shown in <Fig. 23b>. Data means ± SD. Student's t-test. Mice were euthanized 23 days after tumor inoculation. The mice administered with the nanoparticles of the present invention were inoculated with CT26 cells (1x10 ⁵ cells/mouse), and the tumor growth was monitored every 2-3 days after the tumor was palpable and presented as a tumor volume (mm ³ ) < FIG. 23 was equal to c> of Data means ± SD.

<Figure 24> shows the therapeutic efficacy of RNA/DNA1_multi_KRASi@LS, RNA/DNA2_multi_KRASi@LS, and RNA/DNA3_multi_KRASi@LS nanoparticles inoculation in a CT26 tumor model, and the time course of tumor administration and nanoparticle treatment was observed. As shown in <Fig. 25a>, CT26 cells (1x10 ⁵ cells/mouse) were subcutaneously injected into the right flank of 6-8 week old female BalB/c, and 2 days after cell injection, mice (n = 5 -8) was randomly divided, and nanoparticles or PBS were administered to mice in each group three times at an interval of one week. Tumors were monitored every 2-3 days and the results measured using Vernier calipers were as shown in <Fig. 25b>. Tumor growth curves were plotted by measuring tumor volume over time in each group. Data are expressed as mean ± SD.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as including all modifications or chemically modified forms derived from the meaning and scope of the claims to be described later rather than the above detailed description and their equivalent concepts are included in the scope of the present invention.

Claims (29)

A system comprising at least one endogenous protein synthesizing and transcribeable component in a cell, the system comprising:
a DdRp RNA expression cassette comprising DNA dependent RNA Polymerase (DdRp) mRNA with a structure that enables intracellular translation; and
A self-transcribed RNA/DNA system comprising a; a DNAi expression cassette comprising a DNAi encoding an mRNA of interest (mRNAi) transcribed by the DdRp, wherein they are operably linked. The structure of claim 1 , wherein the structure enabling intracellular translation is
A self-transcribed RNA/DNA system comprising a 5'Cap structure or an internal ribosome entry site (IRES) sequence. The method of claim 1, wherein the DdRp RNA expression cassette is
(1) 5'UTR;
(2) RNA encoding the DdRp; and
(3) 3'UTR; self-transcribed RNA/DNA system characterized in that it comprises an RNA. The method according to claim 1 to 3, wherein the DdRp RNA expression cassette is
A self-transcribed RNA/DNA system comprising chemically modified nucleotides (CM). 5. The method of claim 4, wherein the chemically modified nucleotide (CM) is
A self-transcribed RNA/DNA system comprising at least one selected from the group comprising sugar modifications, base modifications, backbone modifications, and lipid modifications. According to claim 1 to 5, wherein the RNA / DNA system for self-transcription of the DdRp mRNA,
In addition, the auto_DdRp expression cassette comprising a promoter to which DdRp binds and a DNA encoding DdRp; and a self-transcribed RNA/DNA system, characterized in that it is operably linked. The method of claim 6, wherein the auto_DdRp expression cassette is
(1) a promoter to which DdRp binds;
(2) 5'UTRs;
(3) a DNA fragment encoding the DdRp; and
(4) 3'UTRs; A self-transcripting RNA/DNA system comprising a DNA comprising, and consisting of, operably linked. The system according to claim 1 , wherein there is a component capable of synthesizing at least one endogenous protein in a cell and capable of transcription, the system comprising:
the DdRp RNA expression cassette; and
A self-transcriptional RNA/DNA system comprising a; The method of claim 1, wherein the DNAi expression cassette is
(1) a promoter to which DdRp binds;
(2) 5'UTRs;
(3) a DNA fragment encoding mRNAi; and
(4) a self-transcripting RNA/DNA system characterized by DNA comprising 3'UTRs; 10. The method according to claim 8 to 9, wherein the DNAi expression cassette and the auto_DdRp expression cassette are
A self-transcripting RNA/DNA system characterized in that it is in more than one gene carrier. 11. The method of claim 1 to 10, wherein the mRNAi is
A self-transcribed RNA/DNA system comprising an ORF encoding any one selected from the group consisting of antigens, antibodies, therapeutic proteins, peptides and fusion proteins. 12. The method of claim 11, wherein the fusion protein is
a polypeptide composed of two proteins or protein subunits linked between their ends by a linker peptide; and
A self-transcribed RNA/DNA system, characterized in that any one selected from the group comprising; a polypeptide in which amino acids of two donors are interspersed in a fusion protein. 13. The method of claim 1 to 12, wherein the auto_DdRp expression cassette is
A self-transcribed RNA/DNA system characterized by a DdRp expression loop in which DdRp DNA located downstream of a promoter to which DdRp in the auto_DdRp expression cassette binds by DdRp synthesized from the DdRp RNA expression cassette is repeatedly self-transcribed . 14. The method of claim 1 to 13, wherein the DdRp RNA expression cassette is
G/C content, codon optimization (optimizAtion), UTR modification, poly(A) tail with more than 30 adenosine nucleotides, poly(C) sequence, 5' cap (CAP) structure except m7GpppN and histone-stem-loop A self-transcribed RNA/DNA system characterized by a capped DdRp mRNA comprising at least one selected from nucleotide sequence modification (SM) consisting of a sequence. The method according to claim 14, wherein the 5' cap (CAP) structure except for the m7GpppN is
A self-transcripting RNA/DNA system featuring a chemically modified ARCA-cap (CAP). The method of claim 15, wherein the ARCA-cap (CAP) is
Self-transcribed RNA/DNA system featuring phosphorothioate.. 15. The method of claim 14, wherein the DdRp RNA expression cassette is
Self-transcription RNA / DNA system, characterized in that it further comprises m7GpppN 5' cap (CAP). The method according to claim 1 to 17, wherein the DdRp DNA contained in the auto_DdRp expression cassette and the DNAi contained in the DNAi expression cassette are
From sequence modification (SM) consisting of G/C content, codon optimization (optimizAtion), UTR modification, poly(A) tail with more than 30 adenosine nucleotides, poly(C) sequence, and histone-stem-loop sequence Self-transcribed RNA / DNA system comprising at least one selected. 19. The method of claim 5 to 18, wherein the UTR is
A self-transcribed RNA/DNA system characterized by any one selected from alpha globin UTR and beta globin UTR. 20. The method of claim 5 to 19, wherein the UTR is
A self-transcribed RNA/DNA system characterized in that it introduces a 5'UTR derived from the TOP gene. 21. The method of any one of claims 1 to 20,
Self-transcription RNA/DNA system, characterized in that by delivering the RNA/DNA system, the mRNAi is continuously expressed in the cytoplasm for a long period of time in a nuclear-independent manner. 22. The method of any one of claims 1-21, wherein the RNA/DNA system comprises:
A self-transcribed RNA/DNA system comprising an antibiotic selection expression cassette capable of selecting transfected cells. 23. The method of claim 1-22, wherein the DNAi expression cassette and the auto_DdRp expression cassette are
Linear nucleic acid fragment, circular plasmid, lentivirus, adeno-associated virus, self-transcription RNA / DNA system characterized in that any one selected from the group comprising various forms, including ribonucleoprotein (RNP) complex or a combination thereof . The self-transcribed RNA/DNA system kit according to claim 1 , comprising a DdRp RNA expression cassette and a DNAi expression cassette of a persistent self-transcribed RNA/DNA system of the mRNA of interest in the cytoplasm. 25. The cell or individual according to any one of claims 1 to 24, wherein the RNA/DNA system is transduced with a continuous self-transcripting RNA/DNA system of the mRNA of interest in the cytoplasm. 26. The method of claim 1 to 25, wherein DdRp is
any one selected from the group comprising T7 RNA polymerase, T3 RNA polymerase and SP6 polymerase; or
A self-transcribed RNA/DNA system comprising a chimeric enzyme comprising at least one catalytic domain of DdRp. 27. The method of claim 26, wherein the chimeric enzyme is
at least one catalytic domain of RNA triphosphatase,
at least one catalytic domain of guanylyltransferase,
at least one catalytic domain of N ⁷ -guanine methyltransferase and
A self-transcripting RNA/DNA system comprising a chimeric enzyme comprising at least one catalytic domain of DdRp. 28. The method of claim 26-27, wherein the chimeric enzyme is
At least two catalytic domains selected from the group consisting of the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase, the catalytic domain of N ⁷ -guanine methyltransferase and the catalytic domain of DdRp are linked by a linking peptide. Self-transcription RNA / DNA system, characterized in that. 29. The method according to claim 1 to 28, wherein the DNAi expression cassette comprises DNA directing an enzyme or a catalytic domain capable of adding a 5'Cap structure to mRNA synthesized using a portion of the DNA of the DNAi expression cassette as a transcript. Self-transcription RNA / DNA system, characterized in that.

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